WO2008036061A2 - Enzymes and formulations for broad-specificity decontamination of chemical and biological warfare agents - Google Patents

Enzymes and formulations for broad-specificity decontamination of chemical and biological warfare agents Download PDF

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Publication number
WO2008036061A2
WO2008036061A2 PCT/US2006/013031 US2006013031W WO2008036061A2 WO 2008036061 A2 WO2008036061 A2 WO 2008036061A2 US 2006013031 W US2006013031 W US 2006013031W WO 2008036061 A2 WO2008036061 A2 WO 2008036061A2
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WIPO (PCT)
Prior art keywords
seq
sequence
activity
nucleic acid
polypeptide
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PCT/US2006/013031
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French (fr)
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WO2008036061A3 (en
Inventor
Dan E. Robertson
Toby Richardson
Karen Kustedjo
Gabriel Amitai
Keith Lejeune
Jason Berberich
Jennifer Ann Chaplin
Jessica Sinclair
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Verenium Corporation
Agentase, Llc
Life Science Research Israel Ltd.
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Application filed by Verenium Corporation, Agentase, Llc, Life Science Research Israel Ltd. filed Critical Verenium Corporation
Priority to JP2008535508A priority Critical patent/JP2009517002A/en
Priority to EP06851338A priority patent/EP1928560A2/en
Priority to CA002602185A priority patent/CA2602185A1/en
Priority to IL186399A priority patent/IL186399A0/en
Publication of WO2008036061A2 publication Critical patent/WO2008036061A2/en
Publication of WO2008036061A3 publication Critical patent/WO2008036061A3/en

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    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/02—Chemical warfare substances, e.g. cholinesterase inhibitors

Abstract

The invention provides novel enzymes and methods for enzyme-based active decontamination, e.g., nerve agent detoxification. In one aspect, the invention provides enzymes having non-heme chloroperoxidase (nhCPOs) activity, and methods for bleaching and degradation of lignin. The invention provides polypeptides, nucleic acids, infective vehicles, transduced or infected cells or plants and/or transgenic plants to, and methods of using them, e.g., provide self-protecting, pesticide-resistant and/or herbicide-resistant cells or plants, where in one aspect the polypeptides of the invention act to hydrolyzing P-S or P-F bonds and detoxify acetylcholinesterases or butyrylcholinesterases. In one aspect, the invention provides an enzyme mixture or composition that provides broad-spectrum, agent-specific oxidation and hydrolysis activities as well as hypohalite generation for oxidation or hydrolysis of V-agents, G-agents and H-agents and which generates oxidants such as chlorine dioxide and reactive radicals for killing of biological agents, including anthrax spores.

Description

ENZYMES AND FORMULATIONS FOR BROAD- SPECIFICITY DECONTAMINATION OF CHEMICAL AND BIOLOGICAL WARFARE AGENTS
FEDERAL FUNDING LEGEND
This invention was produced in part using funds from the Federal government under Defense Threat Reduction Agency contract # HDTRA-04-C-0046. Accordingly, the Federal government has certain rights in this invention.
TECHNICAL FIELD This invention relates to molecular and cellular biology and biochemistry. In one aspect, the invention provides enzymes and methods for enzyme-based active decontamination, e.g., nerve agent detoxification. In one aspect, the invention provides enzymes having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non- heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, and methods of making and using them, including various industrial applications such as bleaching and degradation of lignin, in addition to their use as decontamination agents. The invention provides polypeptides, nucleic acids, infective vehicles and methods of using them to generate self-protecting, pesticide-resistant cells or plants. In one aspect, the invention provides an enzyme mixture or composition that provides broad-spectrum, agent-specific oxidation and hydrolysis activities as well as hypohalite generation for oxidation or hydrolysis of V-agents, G-agents and H-agents and which generates oxidants such as chlorine dioxide and reactive radicals for killing of biological agents, including anthrax spores.
BACKGROUND
Most insecticides and agaricides used in commercial agriculture are acetyl- or butyrylcholinesterase inhibitors with P-S or P-F bonds as a critical determinant of inhibitory action. These molecules, while toxic to pests can also be toxic to the plants they are applied to. SUMMARY
The invention provides novel polypeptides (e.g., enzymes, peptides and antibodies) and methods for use in enzyme-based active decontamination, e.g., of toxins and poisons, including synthetic substances such as nerve gases, e.g. agent VX, mustard gases and the like, and toxic biological agents, e.g., Bacillus and other toxic spores. The invention provides novel polypeptides, for example, enzymes and catalytic antibodies, having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, and mixtures thereof (e.g., as formulations), for decontamination activity, including thermostable and thermotolerant forms of these enzymes (e.g., having oxidoreductase, hydrolase, etc.) activities, and polynucleotides encoding these polypeptides, including vectors, host cells, transgenic plants and non-human animals, and methods for making and using these polynucleotides and polypeptides.
The invention provides isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues, wherein the nucleic acid encodes at least one polypeptide having at least a hydrolase activity, an esterase activity, e.g., a carboxylesterase activity, an organophosphohydrolase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, and/or a decontamination activity (e.g., pesticides, herbicides, insecticides, and other toxic agents) or other related activity. The sequence identities can be determined by analysis with a sequence comparison algorithm or by a visual inspection. Exemplary nucleic acids of the invention include isolated, synthetic or recombinant nucleic acids comprising a nucleic acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:185, SEQ ID NO: 187, SEQ ID NO:189, SEQ ID NO:191 and SEQ ID NO: 193, and subsequences thereof, e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length, or over the full length of a gene or transcript, and complementary sequences thereof.
Exemplary nucleic acids of the invention also include isolated, synthetic or recombinant nucleic acids encoding a polypeptide of the invention, including exemplary sequences of the invention, e.g., amino acid sequences as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, etc. all the even numbered SEQ ID NO:s through SEQ ID NO: 194, and subsequences thereof and variants thereof. In alternative aspects, the polypeptide has a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non- heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, or a decontamination activity or other related activity. In one aspect, the hydrolase, oxidoreductase, or decontamination activity, or other activity is a regioselective and/or chemoselective activity.
In one aspect, the invention provides polypeptides, including enzymes, peptides and antibodies, and nucleic acids encoding them, wherein the polypeptides have at least one enzymatic activity comprising a detoxifying, neutralizing or decontaminating activity. The enzymatic activity can comprise hydrolysis of, or decontamination of, a V agent. The enzymatic activity can comprise hydrolysis of, or decontamination of, a V agent, or the enzymatic activity can comprises a haloperoxidase activity, or an activity comprising catalyzing the hydrolysis of a methylphosphonofluoridate or a thiophosphoric ester, or a combination thereof. The haloperoxidase activity can comprise a chloroperoxidase activity. The V agent detoxified, neutralized and/or decontaminated by an polypeptide of the invention can comprises VX (0-Ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothioate, or methylphosphonothioic acid), VE (O-Ethyl-S-[2- (diethylamino)ethyl] ethylphosphonothioate), VG (O,O-Diethyl-S-[2- (diethylamino)ethyl] phosphorothioate), VM (0-Ethyl-S-[2-(diethylamino)ethyl] methylphosphonothioate), VR (Phosphonothioic acid) Soviet V-gas (Russian VX), Tetriso (0,0-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate) or a phosphorylthiocholine-comprising compound. The enzyme hydrolyzing or decontaminating a V agent comprises a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 , SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 89; SEQ ID NO:1 17; SEQ ID NO:119; SEQ ID NO: 127; SEQ ID NO: 151; SEQ ID NO: 167; SEQ ID NO: 171; SEQ ID NO: 187 or SEQ ID NO: 191, or having an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:90; SEQ ID NO: 118; SEQ ID NO: 120; SEQ ID NO: 128; SEQ ID NO: 152; SEQ ID NO: 168; SEQ ID NO: 172; SEQ ID NO: 189 or SEQ ID NO: 192.
In one aspect, the invention provides polypeptides, including enzymes, peptides and antibodies, and nucleic acids encoding them, wherein the polypeptides have at least one enzymatic activity comprising a detoxifying, neutralizing or decontaminating activity against a G agent, e.g., the enzymatic activity comprises hydrolysis of, or decontamination of, a G agent. The enzymatic activity can comprise hydrolysis of, or decontamination of, a G agent comprises an organophosphoric acid anhydrolase (OPAA) activity. The G agent can comprise tabun (GA), sarin (methylphosphonofluoridic acid) (GB), soman (GD), cyclosarin (GF) or a combination thereof. The enzyme that hydrolyzes or decontaminates a G agent can comprise a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO:73; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO: 101 ; SEQ ID NO: 103; SEQ ID NO: 105; SEQ ID NO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ ID NO:127;SEQIDNO:129;SEQIDNO:131;SEQIDNO:133;SEQIDNO:135;SEQID NO:137; SEQIDNO:139; SEQIDNO:141; SEQIDNO:143; SEQIDNO:145; SEQID NO:147; SEQ IDNO:149; SEQIDNO:151; SEQ IDNO:153; SEQ IDNO:155; SEQ ID NO:157; SEQIDNO:159; SEQIDNO:161; SEQIDNO:163; SEQIDNO:165; SEQID NO:167; SEQIDNO:169; SEQIDNO:171; SEQIDNO:173; SEQIDNO:175; SEQID NO:177;SEQIDNO:179;SEQIDNO:181;SEQIDNO:183;SEQIDNO:185;SEQID NO: 187 or SEQ ID NO: 191; or has an amino acid sequence as set forth in SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO: 100; SEQ ID NO: 102; SEQ ID NO: 104; SEQ ID NO:106; SEQ ID NO: 108; SEQ ID NO: 110; SEQ ID NO: 112; SEQ ID NO: 114; SEQ ID NO: 116; SEQ ID NO: 118; SEQID NO: 120; SEQ ID NO: 122; SEQ ID NO: 124; SEQ ID NO: 126; SEQ ID NO: 128; SEQ ID NO: 130; SEQ ID NO: 132; SEQ ID NO: 134; SEQ ID NO: 136; SEQ ID NO: 138; SEQID NO: 140; SEQ ID NO: 142; SEQ ID NO: 144; SEQ ID NO: 146; SEQ ID NO: 148; SEQ ID NO:150; SEQIDNO:152; SEQIDNO:154; SEQIDNO:156; SEQIDNO:158; SEQID NO: 160; SEQ ID NO: 162; SEQ ID NO: 164; SEQ ID NO: 166; SEQ ID NO: 168; SEQ ID NO: 170; SEQ ID NO: 172; SEQ ID NO: 174; SEQ ID NO: 176; SEQ ID NO: 178; SEQID NO: 180; SEQ ID NO: 182; SEQ ID NO: 184; SEQ ID NO: 186; SEQ ID NO: 188, SEQ ID NO: 192, or, a polypeptide having a prolidase activity or an organophosphoric acid anhydrolase (OPAA) activity and encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having a sequence as set forth in SEQ ID NO: 194. In one aspect, the invention provides polypeptides, including enzymes, peptides and antibodies, and nucleic acids encoding them, wherein the polypeptides have at least one enzymatic activity comprising a detoxifying, neutralizing or decontaminating activity against an H agent, e.g., the enzymatic activity comprises hydrolysis of, or decontamination of, an H agent. The H agent can comprise a chloroperoxidase activity, a dehalogenase activity or a combination thereof. The H agent can comprise mustard gas; 1 I' thiobis [2 chloroethane] bis-(2-chloroethyl) sulphide; beta, beta1 dichloroethyl sulphide; 2, 2' dichloroethyl sulphide; bis (beta-chloroethyl) sulphide; l-chloro-2 (beta- chlorodiethylthio) ethane; or, a combination thereof. The enzyme hydrolyzing or decontaminating an H agent can comprise a polypeptide encoded by a nucleic acid encoding a chloroperoxidase and having a sequence as set forth in SEQ ID NO:1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively, or a nucleic acid encoding a dehalogenase and having a sequence as set forth in SEQ ID NO: 69 or SEQ ID NO:91 or the enzyme has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92..
In one aspect, the invention provides polypeptides, including enzymes, peptides and antibodies, and nucleic acids encoding them, wherein the polypeptides have at least one enzymatic activity comprising a detoxifying, neutralizing or decontaminating activity against a biological agent, e.g., the enzymatic activity comprises hydrolysis of, or decontamination of, a biological agent. The enzymatic activity can comprise a chloroperoxidase activity. The biological agent can be a gram negative spore, such as a Bacillus spore, e.g., an anthrax, or Bacillus anthracis, spore. The enzyme hydrolyzing, neutralizing or decontaminating a biological agent can comprise a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO:1, SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID
NO:62; SEQ ID NO.64; SEQ ID NO:66 or SEQ ID NO:68, respectively. The enzymatic activity can comprise hydrolysis of, or decontamination of, a P-F bond. The polypeptide having P-F bond hydrolyzing, neutralizing or decontamination activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO: 101; SEQ ID
NO: 103; SEQ ID NO: 105; SEQ ID NO: 107; SEQ ID NO: 109; SEQ ID NO: 111; SEQ ID NO: 113; SEQ ID N0:115; SEQ ID NO: 117; SEQ ID NO: 119; SEQ ID NO: 121; SEQ ID NO: 123; SEQ ID NO: 125; SEQ ID NO: 127; SEQ ID NO: 129; SEQ ID NO: 131; SEQ ID NO: 133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO: 143; SEQ ID NO: 145; SEQ ID NO: 147; SEQ ID NO: 149; SEQ ID NO: 151; SEQ ID
NO: 153; SEQ ID NO:155; SEQ ID NO:157; SEQ ID NO:159; SEQ ID NO:161; SEQ ID
NO:163; SEQ ID NO:165; SEQ ID NO:167; SEQ ID NO:169; SEQ ID NO:171; SEQ ID
NO:173; SEQ ID NO:175; SEQ ID NO: 177; SEQ ID NO:179; SEQ ID NO:181; SEQ ID NO: 183; SEQ ID NO: 185; SEQ ID NO: 187, SEQ ID NO: 191 or SEQ ID NO: 193, or has an amino acid sequence as set forth in SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:94;
SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO: 100; SEQ ID NO: 102; SEQ ID NO: 104;
SEQ ID NO: 106; SEQ ID NO: 108; SEQ ID NO: 110; SEQ ID NO: 112; SEQ ID NO: 114;
SEQ ID NO: 116; SEQ ID NO: 118; SEQ ID NO: 120; SEQ ID NO: 122; SEQ ID NO: 124; SEQ ID NO:126; SEQ ID NO:128; SEQ ID NO: 130; SEQ ID NO:132; SEQ ID NO:134;
SEQ ID NO: 136; SEQ ID NO: 138; SEQ ID NO: 140; SEQ ID NO: 142; SEQ ID NO: 144;
SEQ ID NO: 146; SEQ ID NO: 148; SEQ ID NO: 150; SEQ ID NO: 152; SEQ ID NO: 154;
SEQ ID NO: 156; SEQ ID NO: 158; SEQ ID NO: 160; SEQ ID NO: 162; SEQ ID NO: 164;
SEQ ID NO: 166; SEQ ID NO: 168; SEQ ID NO: 170; SEQ ID NO: 172; SEQ ID NO: 174; SEQ ID NO: 176; SEQ ID NO: 178; SEQ ID NO: 180; SEQ ID NO: 182; SEQ ID NO: 184;
SEQ ID NO: 186; SEQ ID NO: 188, SEQ ID NO: 192 or SEQ ID NO: 194.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises hydrolysis of, or decontamination of, a P-S bond. The polypeptide having P-S bond hydrolyzing, neutralizing or decontamination activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:1; SEQ ID NO: 75; SEQ ID NO:
77; SEQ ID NO: 89; SEQ ID NO: 117; SEQ ID NO: 119; SEQ ID NO: 127; SEQ ID
NO: 151; SEQ ID NO:167; SEQ ID NO:171; SEQ ID NO:187 or SEQ ID NO:191, or has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:76; SEQ ID NO: 78; SEQ ID NO:90; SEQ ID NO: 118; SEQ ID NO:120; SEQ ID NO: 128; SEQ ID NO: 152;
SEQ ID NO: 168; SEQ ID NO: 172; SEQ ID NO: 188 or SEQ ID NO: 192.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises an organophosphoric acid anhydrolase (OPAA) activity. The enzyme having organophosphoric acid anhydrolase (OPAA) activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or has an amino acid sequence as set forth in SEQ ID NO: 194.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises an aminopeptidase activity. The enzyme having aminopeptidase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 189, or has an amino acid sequence as set forth in SEQ ID NO: 190.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises a carboxylesterase activity. The enzyme having carboxylesterase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87 or SEQ ID NO:89. In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises a heme-based peroxidase activity. The enzyme having heme-based peroxidase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO: 13; SEQ ID NO:15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41 ; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has a sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO: 52, respectively. The enzyme can have a heme-based chloroperoxidase activity and can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 , or has an amino acid sequence as set forth in SEQ ID NO:2.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises a non-heme-based peroxidase activity. The enzyme having non- heme-based chloroperoxidase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises a dehalogenase activity. The enzyme having a dehalogenase activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises a diisopropylfluorophosphatase (DFPase) activity. The enzyme having diisopropylfluorophosphatase (DFPase) activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72. In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises an organophosphoesterase and can hydrolyze a P-S or a P-F bond and detoxify an acetylcholinesterase- or butyrylcholinesterase- inhibitor.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises a halogenation reaction to form a hypohalite from hydrogen peroxide and chloride, bromide or iodide. In one aspect, the enzymatic activity comprises catalysis of the transfer of oxygen from hydrogen peroxide to an organic substrate. In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises chemical bleaching of lignin. The enzymatic activity can comprise chemical bleaching of lignin in a pulp or paper manufacturing process.
In one aspect, the enzymatic activity of a polypeptide of the invention, or a polypeptide used to practice a method of the invention or used in a formulation of the invention, comprises detoxifying a pesticide, herbicide and/or insecticide. The pesticide can comprise Demeton-S, Demeton-S-methyl, Demeton-S-methylsulphon, Demeton- methyl, Parathion, Phosmet, Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos- ethyl, Amiton, Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton, Endothion, Etion, Ethoate-methyl, Formothion, Malathion, Mercarbam, Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone, Prothidathion, Prothoate, Sophamide, Thiometon, Vamidothion, Methamidophos or a combination thereof.
In one aspect, the invention also provides enzyme-encoding nucleic acids with a common novelty in that they are derived from mixed cultures. The invention provides enzyme-encoding nucleic acids isolated from mixed cultures comprising a nucleic acid of the invention, e.g., a nucleic acid having a sequence at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention over a region of at least about 10, 20, 30, 40, 50, 60, 70, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues, wherein the nucleic acid encodes at least one polypeptide having a enzyme activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection. In one aspect, the invention provides enzyme-encoding nucleic acids isolated from mixed cultures comprising a nucleic acid of the invention.
In one aspect, the invention also provides enzyme-encoding nucleic acids with a common novelty in that they are derived from environmental sources, e.g., mixed environmental sources. In one aspect, the invention provides enzyme-encoding nucleic acids isolated from environmental sources, e.g., mixed environmental sources, comprising a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues, wherein the nucleic acid encodes at least one polypeptide having a enzyme activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection. In one aspect, the invention provides enzyme-encoding nucleic acids isolated from environmental sources, e.g., mixed environmental sources, comprising a nucleic acid of the invention. In one aspect, the sequence comparison algorithm is a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d "nr pataa" -F F, and all other options are set to default.
Another aspect of the invention is an isolated, synthetic or recombinant nucleic acid including at least 10 consecutive bases of a nucleic acid sequence of the invention, sequences substantially identical thereto, and the sequences complementary thereto.
In one aspect, the isolated, synthetic or recombinant nucleic acid encodes a polypeptide having a enzyme activity, which is thermostable. The polypeptide can retain activity under conditions comprising a temperature range of between about 370C to about 95°C; between about 550C to about 85°C, between about 7O0C to about 95°C, or, between about 900C to about 95°C.
In another aspect, the isolated or recombinant nucleic acid encodes a polypeptide having an enzyme, structural or binding activity, which is thermotolerant. The polypeptide can retain activity after exposure to a temperature in the range from greater than 37°C to about 950C or anywhere in the range from greater than 55°C to about 85°C. The polypeptide can retain activity after exposure to a temperature in the range between about TC to about 50C, between about 5°C to about 15°C, between about 15°C to about 25°C, between about 25°C to about 37°C, between about 370C to about 95°C, between about 55°C to about 85°C, between about 700C to about 750C, or between about 9O0C to about 950C, or more. In one aspect, the polypeptide retains activity after exposure to a temperature in the range from greater than 900C to about 950C at about pH 4.5. The invention provides isolated, synthetic or recombinant nucleic acids comprising a sequence that hybridizes under stringent conditions to a nucleic acid of the invention, e.g., an exemplary nucleic acid of the invention comprising a sequence (or its complement) as set forth in SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, etc. (all of the odd-numbers SEQ ID NO:s set forth in the sequence listing, i.e., SEQ ID NO: 1 through SEQ ID NO: 193), including fragments or subsequences thereof, and complementary sequences thereof. In one aspect, the nucleic acid encodes a polypeptide having an enzyme activity, e.g., a decontamination activity, including having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity. The nucleic acid can be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length or the full length of the gene or transcript. In one aspect, the stringent conditions include a wash step comprising a wash in 0.2X SSC at a temperature of about 650C for about 15 minutes. The invention provides a nucleic acid probe, e.g., a probe for identifying a nucleic acid encoding a polypeptide having a enzyme activity, wherein the probe comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more, consecutive bases of a sequence of the invention, or fragments or subsequences thereof, wherein the probe identifies the nucleic acid by binding or hybridization. The probe can comprise an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases of a sequence comprising a sequence of the invention, or fragments or subsequences thereof. The probe can comprise an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases of a nucleic acid sequence of the invention, or a subsequence thereof.
The invention provides an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide having an enzyme activity, wherein the primer pair is capable of amplifying a nucleic acid comprising a sequence of the invention, or fragments or subsequences thereof. One or each member of the amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of the sequence. The invention provides methods of amplifying a nucleic acid encoding a polypeptide having enzyme activity, comprising amplification of a template nucleic acid with an amplification primer sequence pair capable of amplifying a nucleic acid sequence of the invention, or fragments or subsequences thereof.
The invention provides expression cassettes comprising a nucleic acid of the invention or a subsequence thereof. In one aspect, the expression cassette can comprise the nucleic acid that is operably linked to a promoter. The promoter can be a viral, bacterial, mammalian or plant promoter. In one aspect, the plant promoter can be a potato, rice, corn, wheat, tobacco or barley promoter. The promoter can be a constitutive promoter. The constitutive promoter can comprise CaMV35S. In another aspect, the promoter can be an inducible promoter. In one aspect, the promoter can be a tissue- specific promoter or an environmentally regulated or a developmentally regulated promoter. Thus, the promoter can be, e.g., a seed-specific, a leaf-specific, a root-specific, a stem-specific or an abscission-induced promoter. In one aspect, the expression cassette can further comprise a plant or plant virus expression vector.
The invention provides cloning vehicles comprising an expression cassette (e.g., a vector) of the invention or a nucleic acid of the invention. The cloning vehicle can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome. The viral vector can comprise an adenovirus vector, a retroviral vector or an adeno -associated viral vector. The cloning vehicle can comprise a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage Pl -derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).
The invention provides transformed cell comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention, or a cloning vehicle of the invention. In one aspect, the transformed cell can be a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell. In one aspect, the plant cell can be a potato, wheat, rice, corn, tobacco or barley cell.
The invention provides transgenic non-human animals comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention. In one aspect, the animal is a mouse.
The invention provides transgenic plants comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention. The transgenic plant can be a corn plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant or a tobacco plant. The invention provides transgenic seeds comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention. The transgenic seed can be rice, a corn seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a peanut or a tobacco plant seed.
The invention provides an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid of the invention. The invention provides methods of inhibiting the translation of an enzyme message in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid of the invention.
The invention provides an isolated, synthetic or recombinant polypeptide comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) sequence identity to an exemplary polypeptide or peptide of the invention over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more residues, or over the full length of the polypeptide, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection. Exemplary polypeptide or peptide sequences of the invention include SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO: 1 10, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO:146, SEQ ID NO.148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO: 158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192 and SEQ ID NO:194, and subsequences thereof and variants thereof, e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length, or over the full length of an enzyme. Exemplary polypeptide or peptide sequences of the invention include sequence encoded by a nucleic acid of the invention. Exemplary polypeptide or peptide sequences of the invention include polypeptides or peptides specifically bound by an antibody of the invention. In one aspect, a polypeptide of the invention has at least one enzyme activity. In one aspect, the activity is a regioselective and/or chemoselective activity.
Another aspect of the invention is an isolated, synthetic or recombinant polypeptide or peptide including at least 10 consecutive bases of a polypeptide or peptide sequence of the invention, sequences substantially identical thereto, and the sequences complementary thereto.
In one aspect, the enzyme activity can be thermostable. The polypeptide can retain an enzyme activity under conditions comprising a temperature range of between about 37°C to about 95°C, between about 55°C to about 85°C, between about 700C to about 95°C, or between about 900C to about 95°C. In another aspect, the enzyme activity can be thermotolerant. The polypeptide can retain an enzyme activity after exposure to a temperature in the range from greater than 37°C to about 950C, or in the range from greater than 550C to about 85°C. In one aspect, the polypeptide can retain an enzyme activity after exposure to a temperature in the range from greater than 9O0C to about 95°C at pH 4.5.
In one aspect, the isolated, synthetic or recombinant polypeptide can comprise the polypeptide of the invention that lacks a signal sequence. In one aspect, the isolated or recombinant polypeptide can comprise the polypeptide of the invention comprising a heterologous signal sequence, such as a heterologous enzyme or non-enzyme signal sequence. In one aspect, the invention provides chimeric proteins comprising a first domain comprising a signal sequence of the invention and at least a second domain. The protein can be a fusion protein. The second domain can comprise an enzyme. The enzyme can be an enzyme (e.g., an enzyme of the invention, or, another enzyme). In one aspect, the enzyme activity comprises a specific activity at about 37°C in the range from about 100 to about 1000 units per milligram of protein. In another aspect, the enzyme activity comprises a specific activity from about 500 to about 750 units per milligram of protein. Alternatively, the enzyme activity comprises a specific activity at 370C in the range from about 500 to about 1200 units per milligram of protein. In one aspect, the enzyme activity comprises a specific activity at 37°C in the range from about 750 to about 1000 units per milligram of protein. In another aspect, the thermotolerance comprises retention of at least half of the specific activity of the enzyme at 370C after being heated to the elevated temperature. Alternatively, the thermotolerance can 5 comprise retention of specific activity at 37°C in the range from about 500 to about 1200 units per milligram of protein after being heated to the elevated temperature.
The invention provides the isolated, synthetic or recombinant polypeptide of the invention, wherein the polypeptide comprises at least one glycosylation site. In one aspect, glycosylation can be an N-linked glycosylation. In one aspect, the polypeptide o can be glycosylated after being expressed in a P. pas (oris or a S. pombe.
In one aspect, the polypeptide can retain an enzyme activity under conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another aspect, the polypeptide can retain an enzyme activity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11. 5 The invention provides protein preparations comprising a polypeptide of the invention, wherein the protein preparation comprises a liquid, a solid or a gel.
The invention provides heterodimers comprising a polypeptide of the invention and a second domain. In one aspect, the second domain can be a polypeptide and the heterodimer can be a fusion protein. In one aspect, the second domain can be an epitope0 or a tag. In one aspect, the invention provides homodimers comprising a polypeptide of the invention.
The invention provides immobilized polypeptides having an enzyme activity, wherein the polypeptide comprises a polypeptide of the invention, a polypeptide encoded by a nucleic acid of the invention, or a polypeptide comprising a polypeptide of the5 invention and a second domain. In one aspect, the polypeptide can be immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.
The invention provides arrays comprising an immobilized nucleic acid of the invention. The invention provides arrays comprising an antibody of the invention.0 The invention provides isolated, synthetic or recombinant antibodies that specifically bind to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention. The antibody can be a monoclonal or a polyclonal antibody. The invention provides hybridomas comprising an antibody of the invention, e.g., an antibody that specifically binds to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention.
The invention provides food supplements for an animal comprising a polypeptide of the invention, e.g., a polypeptide encoded by the nucleic acid of the invention. In one aspect, the polypeptide in the food supplement can be glycosylated. The invention provides edible enzyme delivery matrices comprising a polypeptide of the invention, e.g., a polypeptide encoded by the nucleic acid of the invention. In one aspect, the delivery matrix comprises a pellet. In one aspect, the polypeptide can be glycosylated. In one aspect, the enzyme activity is thermotolerant. In another aspect, the enzyme activity is thermostable.
The invention provides method of isolating or identifying a polypeptide having an enzyme activity comprising the steps of: (a) providing an antibody of the invention; (b) providing a sample comprising polypeptides; and (c) contacting the sample of step (b) with the antibody of step (a) under conditions wherein the antibody can specifically bind to the polypeptide, thereby isolating or identifying a polypeptide having an enzyme activity.
The invention provides methods of making an anti-enzyme antibody comprising administering to a non-human animal a nucleic acid of the invention or a polypeptide of the invention or subsequences thereof in an amount sufficient to generate a humoral immune response, thereby making an anti-enzyme antibody. The invention provides methods of making an anti-enzyme immune comprising administering to a non-human animal a nucleic acid of the invention or a polypeptide of the invention or subsequences thereof in an amount sufficient to generate an immune response.
The invention provides methods of producing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid of the invention operably linked to a promoter; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide. In one aspect, the method can further comprise transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell.
The invention provides methods for identifying a polypeptide having an enzyme activity comprising the following steps: (a) providing a polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the invention; (b) providing an enzyme substrate; and (c) contacting the polypeptide or a fragment or variant thereof of step (a) with the substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of a reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having an enzyme activity. The invention provides methods for identifying an enzyme substrate comprising the following steps: (a) providing a polypeptide of the invention; or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test substrate; and (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as an enzyme substrate.
The invention provides methods of determining whether a test compound specifically binds to a polypeptide comprising the following steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid comprises a nucleic acid of the invention, or, providing a polypeptide of the invention; (b) providing a test compound; (c) contacting the polypeptide with the test compound; and (d) determining whether the test compound of step (b) specifically binds to the polypeptide. The invention provides methods for identifying a modulator of an enzyme activity comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test compound; (c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the enzyme, wherein a change in the enzyme activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the enzyme activity. In one aspect, the enzyme activity can be measured by providing an enzyme substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or, an increase in the amount of the substrate or a decrease in the amount of a reaction product. A decrease in the amount of the substrate or an increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of enzyme activity. An increase in the amount of the substrate or a decrease in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of enzyme activity.
The invention provides computer systems comprising a processor and a data storage device wherein said data storage device has stored thereon a polypeptide sequence or a nucleic acid sequence of the invention (e.g., a polypeptide encoded by a nucleic acid of the invention). In one aspect, the computer system can further comprise a sequence comparison algorithm and a data storage device having at least one reference sequence stored thereon. In another aspect, the sequence comparison algorithm comprises a computer program that indicates polymorphisms. In one aspect, the computer system can further comprise an identifier that identifies one or more features in said sequence. The invention provides computer readable media having stored thereon a polypeptide sequence or a nucleic acid sequence of the invention. The invention provides methods for identifying a feature in a sequence comprising the steps of: (a) reading the sequence using a computer program which identifies one or more features in a sequence, wherein the sequence comprises a polypeptide sequence or a nucleic acid sequence of the invention; and (b) identifying one or more features in the sequence with the computer program. The invention provides methods for comparing a first sequence to a second sequence comprising the steps of: (a) reading the first sequence and the second sequence through use of a computer program which compares sequences, wherein the first sequence comprises a polypeptide sequence or a nucleic acid sequence of the invention; and (b) determining differences between the first sequence and the second sequence with the computer program. The step of determining differences between the first sequence and the second sequence can further comprise the step of identifying polymorphisms. In one aspect, the method can further comprise an identifier that identifies one or more features in a sequence. In another aspect, the method can comprise reading the first sequence using a computer program and identifying one or more features in the sequence. The invention provides methods for isolating or recovering a nucleic acid encoding a polypeptide having an enzyme activity from an environmental sample comprising the steps of: (a) providing an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide having an enzyme activity, wherein the primer pair is capable of amplifying a nucleic acid of the invention; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to the amplification primer pair; and, (c) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the environmental sample, thereby isolating or recovering a nucleic acid encoding a polypeptide having an enzyme activity from an environmental sample. One or each member of the amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of a sequence of the invention.
The invention provides methods for isolating or recovering a nucleic acid encoding a polypeptide having an enzyme activity from an environmental sample comprising the steps of: (a) providing a polynucleotide probe comprising a nucleic acid of the invention or a subsequence thereof; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a); (c) combining the isolated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (a); and (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide having an enzyme activity from an environmental sample. The environmental sample can comprise a water sample, a liquid sample, a soil sample, an air sample or a biological sample. In one aspect, the biological sample can be derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell. The invention provides methods of generating a variant of a nucleic acid encoding a polypeptide having an enzyme activity comprising the steps of: (a) providing a template nucleic acid comprising a nucleic acid of the invention; and (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid. In one aspect, the method can further comprise expressing the variant nucleic acid to generate a variant enzyme polypeptide. The modifications, additions or deletions can be introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination thereof. In another aspect, the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair- deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof. In one aspect, the method can be iteratively repeated until an enzyme having an altered or different activity or an altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced. In one aspect, the variant enzyme polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature. In another aspect, the variant enzyme polypeptide has increased glycosylation as compared to the enzyme encoded by a template nucleic acid.
Alternatively, the variant enzyme polypeptide has an enzyme activity under a high temperature, wherein the enzyme encoded by the template nucleic acid is not active under the high temperature. In one aspect, the method can be iteratively repeated until an enzyme coding sequence having an altered codon usage from that of the template nucleic acid is produced. In another aspect, the method can be iteratively repeated until an enzyme gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.
The invention provides methods for modifying codons in a nucleic acid encoding a polypeptide having an enzyme activity to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention encoding a polypeptide having an enzyme activity; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
The invention provides methods for modifying codons in a nucleic acid encoding a polypeptide having an enzyme activity; the method comprising the following steps: (a) providing a nucleic acid of the invention; and, (b) identifying a codon in the nucleic acid of step (a) and replacing it with a different codon encoding the same amino acid as the replaced codon, thereby modifying codons in a nucleic acid encoding an enzyme.
The invention provides methods for modifying codons in a nucleic acid encoding a polypeptide having an enzyme activity to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention encoding an enzyme polypeptide; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non- preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
The invention provides methods for modifying a codon in a nucleic acid encoding a polypeptide having an enzyme activity to decrease its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid of the invention; and (b) identifying at least one preferred codon in the nucleic acid of step (a) and replacing it with a non-preferred or less preferred codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in a host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to decrease its expression in a host cell. In one aspect, the host cell can be a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or a mammalian cell. The invention provides methods for producing a library of nucleic acids encoding a plurality of modified enzyme active sites or substrate binding sites, wherein the modified active sites or substrate binding sites are derived from a first nucleic acid comprising a sequence encoding a first active site or a first substrate binding site the method comprising the following steps: (a) providing a first nucleic acid encoding a first active site or first substrate binding site, wherein the first nucleic acid sequence comprises a sequence that hybridizes under stringent conditions to a nucleic acid of the invention, and the nucleic acid encodes an enzyme active site or an enzyme substrate binding site; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of active site-encoding or substrate binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified enzyme active sites or substrate binding sites. In one aspect, the method comprises mutagenizing the first nucleic acid of step (a) by a method comprising an optimized directed evolution system, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, and a combination thereof. In another aspect, the method comprises mutagenizing the first nucleic acid of step (a) or variants by a method comprising recombination, recursive sequence recombination, phosphothioate-modifϊed DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair- deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
The invention provides methods for making a small molecule comprising the following steps: (a) providing a plurality of biosynthetic enzymes capable of synthesizing or modifying a small molecule, wherein one of the enzymes comprises an enzyme encoded by a nucleic acid of the invention; (b) providing a substrate for at least one of the enzymes of step (a); and (c) reacting the substrate of step (b) with the enzymes under conditions that facilitate a plurality of biocatalytic reactions to generate a small molecule by a series of biocatalytic reactions. The invention provides methods for modifying a small molecule comprising the following steps: (a) providing an enzyme enzyme, wherein the enzyme comprises a polypeptide of the invention, or, a polypeptide encoded by a nucleic acid of the invention, or a subsequence thereof; (b) providing a small molecule; and (c) reacting the enzyme of step (a) with the small molecule of step (b) under conditions that facilitate an enzymatic reaction catalyzed by the enzyme enzyme, thereby modifying a small molecule by an enzyme enzymatic reaction. In one aspect, the method can comprise a plurality of small molecule substrates for the enzyme of step (a), thereby generating a library of modified small molecules produced by at least one enzymatic reaction catalyzed by the enzyme enzyme. In one aspect, the method can comprise a plurality of additional enzymes under conditions that facilitate a plurality of biocatalytic reactions by the enzymes to form a library of modified small molecules produced by the plurality of enzymatic reactions. In another aspect, the method can further comprise the step of testing the library to determine if a particular modified small molecule which exhibits a desired activity is present within the library. The step of testing the library can further comprise the steps of systematically eliminating all but one of the biocatalytic reactions used to produce a portion of the plurality of the modified small molecules within the library by testing the portion of the modified small molecule for the presence or absence of the particular modified small molecule with a desired activity, and identifying at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.
The invention provides methods for determining a functional fragment of an enzyme comprising the steps of: (a) providing an enzyme enzyme, wherein the enzyme comprises a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, or a subsequence thereof; and (b) deleting a plurality of amino acid residues from the sequence of step (a) and testing the remaining subsequence for an enzyme activity, thereby determining a functional fragment of an enzyme enzyme. In one aspect, the enzyme activity is measured by providing an enzyme substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product. The invention provides methods for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps: (a) making a modified cell by modifying the genetic composition of a cell, wherein the genetic composition is modified by addition to the cell of a nucleic acid of the invention; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis. In one aspect, the genetic composition of the cell can be modified by a method comprising deletion of a sequence or modification of a sequence in the cell, or, knocking out the expression of a gene. In one aspect, the method can further comprise selecting a cell comprising a newly engineered phenotype. In another aspect, the method can comprise culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.
The invention provides methods of increasing thermo tolerance or thermostability of an enzyme polypeptide, the method comprising glycosylating an enzyme polypeptide, wherein the polypeptide comprises at least thirty contiguous amino acids of a polypeptide of the invention; or a polypeptide encoded by a nucleic acid sequence of the invention, thereby increasing the thermotolerance or thermostability of the polypeptide. In one aspect, the enzyme activity can be thermostable or thermotolerant at a temperature in the range from greater than about 37°C to about 95°C.
The invention provides methods for overexpressing a recombinant polypeptide in a cell comprising expressing a vector comprising a nucleic acid comprising a nucleic acid of the invention or a nucleic acid sequence of the invention, wherein the sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection, wherein overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector.
The invention provides detergent compositions comprising a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention, wherein the polypeptide comprises an enzyme activity. In one aspect, the enzyme can be a nonsurface-active enzyme. In another aspect, the enzyme can be a surface-active enzyme.
The invention provides methods for washing an object comprising the following steps: (a) providing a composition comprising a polypeptide having an enzyme activity, wherein the polypeptide comprises: a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing an object; and (c) contacting the polypeptide of step (a) and the object of step (b) under conditions wherein the composition can wash the object.
The invention provides methods of making a transgenic plant comprising the following steps: (a) introducing a heterologous nucleic acid sequence into the cell, wherein the heterologous nucleic sequence comprises a nucleic acid sequence of the invention, thereby producing a transformed plant cell; and (b) producing a transgenic plant from the transformed cell. In one aspect, the step (a) can further comprise introducing the heterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts. In another aspect, the step (a) can further comprise introducing the heterologous nucleic acid sequence directly to plant tissue by DNA particle bombardment. Alternatively, the step (a) can further comprise introducing the heterologous nucleic acid sequence into the plant cell DNA using an Agrobacterium tumefaciens host. In one aspect, the plant cell can be a potato, corn, rice, wheat, tobacco, or barley cell.
The invention provides methods of expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps: (a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence comprises a nucleic acid of the invention; (b) growing the plant under conditions wherein the heterologous nucleic acids sequence is expressed in the plant cell.
The invention provides signal sequences comprising or consisting of a peptide having a subsequence of a polypeptide of the invention, e.g., as described herein. The invention provides a chimeric protein comprising a first domain comprising a signal sequence of the invention and at least a second domain. The protein can be a fusion protein. The second domain can comprise an enzyme. The enzyme can be a hydrolase and/or an oxidoreductase, or enzymes with decontamination activity.
The invention provides methods for decreasing the amount of a compound in a composition comprising the following steps: (a) providing a polypeptide having an enzyme activity or encoding a protein of the invention, or a polypeptide encoded by a nucleic acid of the invention; (b) providing a composition comprising the compound; and (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the enzyme hydrolyzes, dehalogenates, oxidizes, breaks up or otherwise processes the compound in the composition.
The invention provides cellulose-comprising compounds comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, and optionally the cellulose-comprising compound comprises a pulp, a paper, a paper product, a wood, a wood product, or a paper or wood waste product. The invention provides methods for chemical bleaching of lignin or a cellulose in a pulp or paper manufacturing process comprising the steps of (a) providing a polypeptide having chloroperoxidase activity of the invention, or a polypeptide having chloroperoxidase activity encoded by a nucleic acid of the invention; (b) providing a compound comprising a lignin or a cellulose; and (c) contacting the compound with the polypeptide under conditions wherein the polypeptide is enzymatically active and the lignin or a cellulose is bleached, wherein optionally the enzymatic activity comprises a heme-based peroxidase activity, and optionally the enzyme having heme-based peroxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 1 1; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO:21 ; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31 ; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41 ; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51 , or the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO: 50 or SEQ ID NO:52, respectively, and optionally the enzyme has a heme-based chloroperoxidase activity and is encoded by a nucleic having a sequence as set forth in SEQ ID NO:1, or the enzyme has an amino acid sequence as set forth in SEQ ID NO:2, wherein optionally the enzymatic activity comprises a non-heme-based peroxidase activity, and optionally the enzyme having non-heme-based chloroperoxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 ; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively, and optionally the cellulose or lipophilic compound comprises a pulp, a paper, a paper product, a wood, a wood product, or a paper or wood waste product.
The invention provides compositions comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the composition is formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a pill, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof, and optionally the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
The invention provides pharmaceutical compositions comprising a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the pharmaceutical composition is formulated as an edible delivery agent, an injectable liquid, a spray, a tablet, a gel, a liposome, a capsule, a geltab, a hydrogel, a lotion, a topical applied liquid, a suppository, an aerosol, a powder, a lyophilized compound, a propellant, a foam, an emulsion, a nanostructure, an implant or a combination thereof; wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
The invention provides nanostructures (e.g., nanotubules, nanoimplants) comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the nanostructure comprises a nanotubule or a nanofiber, and optionally the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention.
The invention provides lyophilized polypeptides having a sequence of the invention, or a polypeptide encoded by a nucleic acid of the invention. In one aspect, the polypeptide is present in the lyophilate at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or more.
The invention provides decontaminating, neutralizing or detoxifying compositions comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the decontaminating or detoxifying composition is formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof, wherein optionally the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
In one aspect, the decontaminating, neutralizing or detoxifying compositions further comprises a surfactant, an emulsifier, a foaming agent or a combination thereof. The decontaminating, neutralizing or detoxifying composition can be formulated as a pesticide, herbicide and/or insecticide detoxifying agent, a nerve gas detoxifying agent and the like.
In one aspect, when the decontaminating, neutralizing or detoxifying composition comprises a haloperoxidase enzyme, the composition also comprises halite component, and optionally the halite component comprises a chlorite component, and optionally the chlorite component comprises a sodium chlorite or a sodium iodite.
The invention provides products of manufacture comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention. The products of manufacture can comprise a haloperoxidase enzyme, the composition also comprises halite component, and optionally the halite component comprises a chlorite component, and optionally the chlorite component comprises a sodium chlorite or a sodium iodite.
The invention provides cloth, clothing, textiles, threads and/or fibers comprising at least one polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, wherein optionally the at least one polypeptide is immobilized onto the surface of the cloth, textile, thread or fiber, or the at least one polypeptide is a component of a coating or covering on the surface of the cloth, textile or fiber, or a formulation embedded or washed into or onto the cloth, textile or fiber, wherein optionally the composition comprises at least two or three polypeptides of the invention, or a polypeptide encoded by a nucleic acid of the invention.
In one aspect, when the decontaminating, neutralizing or detoxifying composition comprises a mixture of at least two, three, four, five or six different classes of enzymes, wherein optionally each class of enzyme detoxifies a different toxic agent or biological agent. For example, in one aspect, a decontaminating, neutralizing or detoxifying composition of the invention comprises a mixture of at least three different classes of enzymes comprising at least one dehalogenase, at least one haloperoxidase, and at least one organophosphoric acid anhydrolase (OPAA), and optionally the haloperoxidase is a chloroperoxidase. In one aspect, the dehalogenase is a polypeptide having a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively, or the chloroperoxidase is a heme- based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1, or having an amino acid sequence as set forth in SEQ ID NO:2; or the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11;
SEQ ID NO: 13; SEQ ID NO.15; SEQ ID NO: 17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41 ; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively; or the haloperoxidase is a non-heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 ; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO.60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively; or the organophosphoric acid anhydrolase (OPAA) is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having a sequence as set forth in SEQ ID NO: 194. In one aspect, the decontaminating, neutralizing or detoxifying composition comprises a mixture of at least two different classes of enzymes comprising at least one diisopropylfluorophosphatase (DFPase) or organophosphoric acid anhydrolase (OPAA), and at least one haloperoxidase, and optionally the haloperoxidase is a chloroperoxidase. The composition can comprise a diisopropylfluorophosphatase (DFPase), a organophosphoric acid anhydrolase (OPAA) and a haloperoxidase. The DFPase and/or OPAA can be used to decontaminate, neutralize or detoxify G agents, and the haloperoxidase or chloroperoxidase (CPO) can be used to decontaminate, neutralize or detoxify V agents, H agents and/or biological agents, wherein optionally the biological agent is a bacterial spore. In one aspect, this decontaminating, neutralizing or detoxifying composition further comprises a dehalogenase, and/or a cholinesterase. In one aspect, the diisopropyl-fluorophosphatase (DFPase) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72. In one aspect, the haloperoxidase is a chloroperoxidase. In one aspect, the chloroperoxidase is a heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 , or having an amino acid sequence as set forth in SEQ ID NO:2; or the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO: 17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31 ; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively; or the haloperoxidase is a non- heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID
NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively. In one aspect, organophosphoric acid anhydrolase (OPAA) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
In one aspect, the decontaminating, neutralizing or detoxifying composition comprises a mixture of at least two different classes of enzymes comprising at least one dehalogenase (DH) and at least one haloperoxidase, wherein optionally the haloperoxidase is a chloroperoxidase (CPO). The dehalogenases and/or haloperoxidases can be used to decontaminate, neutralize or detoxify H agents. In one aspect, the haloperoxidase is a heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1, or having an amino acid sequence as set forth in SEQ ID NO:2; or the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO:15; SEQ ID NO: 17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID
NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO:14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID
NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively; or the haloperoxidase is a non- heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively. In one aspect of this formulation, the dehalogenase has a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
In one aspect, this formulation further comprises adding at least one diisopropylfluorophosphatase (DFPase), organophosphoric acid anhydrolase (OPAA) and/or cholinesterase to decontaminate, neutralize or detoxify G agents. The diisopropylfluorophosphatase (DFPase) can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71 , or has an amino acid sequence as set forth in SEQ ID NO:72. The organophosphoric acid anhydrolase (OPAA) activity can be encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
The invention provides a decontaminating, neutralizing or detoxifying composition comprising a mixture of at least two different classes of enzymes comprising at least one dehalogenase (DH) enzyme and at least one organophosphoric acid anhydrolase (OPAA). The dehalogenases can be used to decontaminate, neutralize or detoxify an H agent, and the organophosphoric acid anhydrolase can be used to decontaminate, neutralize or detoxify G agents. In one aspect of this formulation, the composition further comprises at least one haloperoxidase to augment the decontamination, neutralization, detoxification of H agents. In one aspect of this formulation, the composition further comprises a haloperoxidase, and optionally the composition can also comprise a halite component, wherein optionally the halite component comprises an iodite or a chlorite component; and optionally the iodite or a chlorite component comprises a sodium chlorite or a sodium iodite or equivalent components. In one aspect of this formulation, the composition further comprises at least one diisopropylfluorophosphatase (DFPase) and/or cholinesterase enzyme to supplement the OPAA decontamination, neutralization or detoxification of a G agent.
The invention provides foaming agents comprising a decontaminating, neutralizing or detoxifying composition of the invention, wherein optionally an enzyme in the foaming agent is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%. The invention provides emulsifying agents or surfactants comprising a decontaminating, neutralizing or detoxifying composition of the invention, wherein optionally an enzyme in the foaming agent or surfactant is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
The invention provides paints or coatings (including undercoats/ undercoatings, boat hull treatments, and the like) comprising a decontaminating, neutralizing or detoxifying composition of the invention, wherein optionally an enzyme in the paint or coating is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
The invention provides methods for decontaminating, neutralizing or detoxifying a toxic agent comprising application or administration of a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition of the invention. The composition used in this method can be formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof. The invention provides methods for preventing the toxic effects of a V agent, an H agent, a G agent or a biological agent comprising application or administration of a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition of the invention.
The invention provides gas masks, air or water filters, and the like, comprising a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition of the invention. These filters can be placed, e.g., in ventilation systems and other air flow systems, e.g., on or in buildings, airplanes, boats, cars, trucks, trains, shelters and the like.
The invention provides a pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agents comprising a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition of the invention, wherein optionally an enzyme in the composition is present at a concentration of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or is present at a w/v of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%. The a pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agents can comprise an organophosphoesterase that hydrolyzes P-S or P-F bonds; and in one aspect, the organophosphoesterase acts as an inhibitor of an acetyl-cholinesterases or a butyrylcholinesterase. In one aspect, the pesticide comprises Demeton-S, Demeton-S- methyl, Demeton-S-methylsulphon, Demeton-methyl, Parathion, Phosmet,
Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos-ethyl, Amiton, Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton, Endothion, Etion, Ethoate- methyl, Formothion, Malathion, Mercarbam, Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone, Prothidathion, Prothoate, Sophamide, Thiometon, Vamidothion, Methamidophos or a combination thereof. The pesticide decontaminating, neutralizing or detoxifying agent can be formulated as or with a coating, a paint, a foam, a liquid, a gel, a lotion, a surfactant, a powder or an emulsifier.
The invention provides methods for decontaminating, neutralizing or detoxifying a pesticide-, herbicide- or insecticide-comprising composition or application, or methods for administration of a decontaminating, neutralizing or detoxifying composition of the invention, or a pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agent of the invention.
The invention provides methods for preventing the toxic effects of a pesticide, herbicide and/or insecticide comprising application or administration of a decontaminating, neutralizing or detoxifying composition of the invention, or a pharmaceutical composition a of the invention, or the pesticide decontaminating, neutralizing or detoxifying agent of the invention.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications, GenBank sequences are hereby expressly incorporated by reference for all purposes.
BRIEF DESCRIPTION OF DRAWINGS
The following drawings are illustrative of aspects of the invention and are not meant to limit the scope of the invention as encompassed by the claims. Figure 1 is a block diagram of a computer system. Figure 2 is a flow diagram illustrating one aspect of a process for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.
Figure 3 is a flow diagram illustrating one aspect of a process in a computer for determining whether two sequences are homologous.
Figure 4 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence.
Figure 5 is an illustration of an anti-CPO antibody western blot of various Pichia and Saccharomyces transformants, as discussed in detail in Example 2, below. Figure 6 illustrates data from a study of tetriso degradation in concentrated sodium chlorite (without any enzyme); Figure 6 A illustrates tetriso levels over time; Figure 6C illustrates chlorite concentration over time; Figure 6D illustrates ClO2 production over time, and Figure 6B illustrates pH over time, as discussed in detail in Example 2, below. Figure 7 illustrates data from a study of bromide ion formation from dehalogenase-catalyzed hydrolysis of dibromomethane, which was assayed as a function of enzyme concentration in various buffers, as discussed in detail in Example 2, below.
Figure 8 illustrates data from a study showing the rate of fluoride produced by DFPase-catalyzed hydrolysis of DFP as a function of DFPase concentration in various buffers, as discussed in detail in Example 2, below.
Figure 9 illustrates data from a study showing the capacity of various buffers to resist pH change after titration with acid, as discussed in detail in Example 2, below.
Figures 10 and 11 illustrate data from a study showing the rate of VX chemical hydrolysis as measured at different NaOH concentrations, i.e., using either 10 μM or 0.5 mM VX, respectively, as discussed in detail in Example 2, below.
Figure 12 illustrate data from a study describing the concentration dependence of BChE inhibition by VX and GF using either RB or butyrylthiocholine (BTC) as substrates, as discussed in detail in Example 2, below.
Figure 13 illustrates studies describing CPO-generated oxidizing radicals concentrations as determined by DPD assay, as discussed in detail in Example 2, below.
Figure 14 illustrates studies describing the efficacy of attenuated B. anthracis spores inactivation by CPO-generated oxidizing radicals, as discussed in detail in Example 2, below. Figures 15A and 15B illustrate data displaying the kinetics of VX degradation by environmental library samples, as discussed in detail in Example 3, below.
Figures 16A and 16B illustrate data displaying the kinetics of Tetriso degradation by these environmental library samples, as discussed in detail in Example 3, below. Figure 17 illustrates data displaying the kinetics of VX degradation by all nine
VX environmental hits compared to a control, as discussed in detail in Example 3, below. Figure 18 illustrates an anti-C fumago CPO immunoblot of various samples of a nucleic acid (SEQ ID NO: 1) encoding a C. fumago chloroperoxidase (CPO) (SEQ ID NO:2) subcloned into Saccharomyces, as discussed in detail in Example 3, below. Figures 19A and 19B illustrate Matrix Assisted Laser Desorption Time of Flight
Mass Spectrometry (MALDI-TOFMS) analyses performed on samples of C. fumago chloroperoxidase (SEQ ID NO:2), as discussed in detail in Example 3, below.
Figures 2OA and 2OB: Figure 2OA illustrates electrospray ionization coupled with a quadrupole time of flight mass spectrometry (ESI-QTOFMS) instrument to perform tandem mass spectrometry to determine the sequence of peptides from the C. fumago chloroperoxidase that were changing in abundance between the untreated and treated samples, as illustrated in Figure 2OA; and peaks which disappeared were selected for tandem mass spectrometry in the untreated sample to determine their sequence, as illustrated in Figure 2OB, as discussed in detail in Example 3, below. Figure 21 illustrates peptides mapped to the CPO 3D structure using electrospray ionization coupled with a quadrupole time of flight mass spectrometry (ESI-QTOFMS) were are on the outside of the molecule, as discussed in detail in Example 3, below.
Figure 22 illustrates data showing Tetriso degradation by various concentrations of CPO in 320 mM chlorite and 300 mM sodium phosphate buffer, as discussed in detail in Example 3, below.
Figure 23 illustrates bromide ion formation from dehalogenase-catalyzed hydrolysis of dibromoethane as a function of enzyme concentration in various buffers, as discussed in detail in Example 3, below.
Figure 24 illustrates the rate of fluoride produced by DFPase catalyzed hydrolysis of DFP as a function of DFPase concentration in various buffers, as discussed in detail in Example 3, below.
Figure 25 illustrates corrosion studies comparing chlorine dioxide with exemplary enzymatic decon formulations (DFPase, Chloroperoxidase, 25 mM sodium chlorite), and shows the enzymatic decontaminant to be less corrosive to metals, as discussed in detail in Example 4, below.
Figure 26 illustrates studies regarding chloroperoxidase and oxidative decontamination, as discussed in detail in Example 4, below. Figure 27 illustrates studies regarding degradation of tetriso by chlorine dioxide and sodium chlorite, as discussed in detail in Example 4, below.
Figure 28 illustrates studies regarding degradation of tetriso by chloroperoxidase, NaClO2 and ClO2, as discussed in detail in Example 4, below.
Figure 29 illustrates studies regarding inactivation of B. subtilis var Niger by sodium chlorite and bioxidation, as discussed in detail in Example 4, below.
Figure 30 illustrates studies demonstrating inactivation of B. subtilis var Niger spores by sodium chlorite and bioxidation using chloroperoxidase or horseradish peroxidase, as discussed in detail in Example 4, below.
Figure 31 illustrates studies demonstrating the stability of chloroperoxidase in buffer, in NaClO2, in NaClO2 with tetriso, and in ClO2, as discussed in detail in Example 4, below.
Figure 32 illustrates studies demonstrating the stability of horseradish peroxidase in buffer, in NaClO2, in NaClO2 with Tetriso, and in ClO2, as discussed in detail in Example 4, below. Figure 33 illustrates studies demonstrating Tetriso degradation by CPO in phosphate buffer with NaClO2, as discussed in detail in Example 4, below.
Figure 34 illustrates studies demonstrating Tetriso degradation by phosphate buffer with NaClO2, as discussed in detail in Example 4, below.
Figure 35 illustrates studies demonstrating Tetriso degradation by CPO phosphate buffer) with 160 mM, as discussed in detail in Example 4, below.
Figure 36 illustrates studies demonstrating a decrease in ClO2 absorbance upon the addition of CPO, as discussed in detail in Example 4, below.
Figure 37 illustrates studies demonstrating the kinetics of ClO2 degradation upon addition of CPO or an equivalent weight of BSA, as discussed in detail in Example 4, below.
Figure 38 illustrates two photos of blue-tempered spring steel by enzyme-based decontamination solution (left panel) and 340 ppm chlorine dioxide (right panel), as discussed in detail in Example 4, below. Figure 39 illustrates studies demonstrating the tm for sulfur mustard (HD) degradation by the exemplary enzyme of the invention SEQ ID NO:70, as discussed in detail in Example 4, below.
Figure 40 illustrates data showing the residual percent sulfur mustard (HD) following enzymatic degradation by the exemplary SEQ ID NO: 70 at various enzyme (DHG) concentrations, as discussed in detail in Example 4, below.
Figure 41 illustrates data showing the residual percent sulfur mustard (HD) following enzymatic degradation by the exemplary SEQ ID NO: 70 at various sulfur mustard (HD) concentrations, as discussed in detail in Example 4, below. Figure 42 illustrates data showing the degradation of sulfur mustard (HD by the exemplary SEQ ID NO:70 with polyethyleneglycol-400 (PEG-400), as discussed in detail in Example 4, below.
Figure 43 illustrates data displaying that CPOZNaClO2 causes >99% degradation of sulfur mustard (HD) within 1 minute (min), and illustrates data displaying the time- dependence of HD degradation by the exemplary SEQ ID NO: 70, as discussed in detail in Example 4, below.
Figure 44 illustrates data showing the degradation of HD by the exemplary SEQ ID NO:70 at various conditions and times, as discussed in detail in Example 4, below.
Figure 45 illustrates data showing the exemplary dehalogenase (DHG) SEQ ID NO:70 was an active hydrolyzing enzyme for degradation of HD-SO, and illustrates the time course of_enzymatic degradation of HD-SO by the exemplary SEQ ID NO: 70, at various conditions and times, as discussed in detail in Example 4, below.
Figure 46 illustrates the time-course of VX degradation by C. fitmago chloroperoxidase (CPO) (SEQ ID NO:2) at low activity level, Figure 46A shows data generated at VX 10 μM, NaClO2 0.03 M; Figure 46B shows data generated at VX 1 μM, NaClO2 0.02 M, Phosphate 5OmM, pH 7.5, 250C, as discussed in detail in Example 4, below.
Figure 47 describes the kinetics of VX degradation measured in 96-well plates containing lysate samples at specified time intervals, as discussed in detail in Example 4, below.
Figure 48 illustrates data showing "Tetriso hits" (enzymes that hydrolyze Tetriso) discovered by the robotic screening assay as discussed in detail in Example 4, below. Figure 49 and Figure 50 summarize data of all VX and Tetriso degradation kinetics for all library "hits" discovered by the robotic screening assay as discussed in detail in Example 4, below.
Figure 51 summarizes data indicating that VX oxidation by CPO/NaClCh as well as GF (cyclohexyl sarin) and DFP hydrolysis by DFPase in 0.2 M phosphate proceed to completion at a relatively rapid rate, whereas VX degradation in 0.2 M AC is slow and incomplete, as discussed in detail in Example 4, below.
Figure 52 illustrates photos of carbon steel exposed to levels of chlorine dioxide "normally" generated when practicing and using these haloperoxidase/ chlorite component decontamination (decon) formulations of the invention, as discussed in detail in Example 4, below.
Figure 53 illustrates an exemplary study showing that the exemplary CPO of the invention is effective in decontaminating bacterial spores, as discussed in detail in Example 4, below.
Figure 54 illustrates data from a time-course detoxification study using an exemplary OPAA enzyme of the invention, as discussed in detail in Example 7, below.
Figure 55 illustrates data from a gas chromatograph analysis of the time- course detoxification study illustrated in Figure 54, as discussed in detail in Example 7, below.
Figure 56 illustrates data from a time-course detoxification study of HD using an exemplary dehalogenase of the invention, as discussed in detail in Example 8, below. Figure 57 illustrates data from a time-course detoxification study of VX using an exemplary CPO enzyme of the invention, as discussed in detail in Example 9, below.
Like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION The invention provides polypeptides, including peptides, enzymes and antibodies, having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention provides mixtures/ formulations/ combinations of enzymes of the invention, and in some aspects, these mixtures/ formulations/ combinations also comprise known enzymes, for decontamination or detoxification (including neutralization) activity. In one aspect, the invention is directed to polypeptides, e.g., enzymes, having any of these activities (e.g., hydrolase, dehalogenase, haloperoxidase, oxidoreductase activity, organophosphoric acid anhydrolase, organophosphohydrolase, etc.) including enzymes having thermostable and thermotolerant enzyme activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides.
The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts, including active decontamination. In another aspect, the polypeptides of the invention are used to synthesize enantiomerically pure chiral products. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts, including active decontamination. For example, in one aspect, the invention provides polypeptides (including enzymes, peptides, antibodies of the invention), and novel mixture/ formulation/ combinations of enzymes (including polypeptides of the invention, known enzymes, or a mixture thereof) for decontamination or detoxification (which includes neutralization), e.g., of toxins such as nerve agents, e.g., V agents (VX agent), G agents (sarin, soman, cyclosarin) or H agents (e.g., mustard gases) and/or biological agents (e.g., anthrax spores), for civilian, military and/or homeland security purposes. Enzymes of the invention can be highly selective catalysts. They can have the ability to catalyze reactions with stereo-, regio-, and chemo- selectivities not possible in conventional synthetic chemistry. Enzymes of the invention can be versatile. In various aspects, they can function in organic solvents, operate at extreme pHs (for example, high pHs and low pHs) extreme temperatures (for example, high temperatures and low temperatures), extreme salinity levels (for example, high salinity and low salinity), and catalyze reactions with compounds that are structurally unrelated to their natural, physiological substrates. Generating and Manipulating Nucleic Acids
The invention provides nucleic acids, including expression cassettes such as expression or cloning vectors, encoding the polypeptides (e.g., enzymes, peptides, antibodies) of the invention. The invention also includes methods for discovering new enzyme sequences using the nucleic acids of the invention. Also provided are methods for modifying the nucleic acids of the invention by, e.g., synthetic ligation reassembly, optimized directed evolution system and/or saturation mutagenesis. Also provided are nucleic acids capable of inhibiting expression of enzymes, including for example antisense or RNAi (double-stranded "interfering" RNA, including miRNA or iRNA) having a sequence of the invention.
The nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. In practicing the methods of the invention, homologous genes can be modified by manipulating a template nucleic acid, as described herein. The invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.
General Techniques
The nucleic acids used to practice this invention, whether RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems. Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066.
Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), VoIs. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Patent Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); Pl artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; Pl-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120- 124; cosmids, recombinant viruses, phages or plasmids.
In one aspect, a nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof. The invention provides fusion proteins and nucleic acids encoding them. A polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification. Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like. Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine- tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego CA) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification. For example, an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-414). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
The term "isolated" can mean that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. As used herein, an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition. Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity. In alternative aspects, the invention provides nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.
The term "recombinant" can mean that the nucleic acid is adjacent to a "backbone" nucleic acid to which it is not adjacent in its natural environment. In one aspect, nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid "backbone molecules." "Backbone molecules" according to the invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest. In one aspect, the enriched nucleic acids represent 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. "Recombinant" polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein. "Synthetic" polypeptides or protein are those prepared by chemical synthesis, as described in further detail, below. A "coding sequence of or a "sequence encodes" a particular polypeptide or protein, is a nucleic acid sequence which is transcribed and translated into a polypeptide or protein when placed under the control of appropriate regulatory sequences.
The term "gene" can include a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability. Genes can include, inter alia, regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons). The phrases "nucleic acid" or "nucleic acid sequence" can include an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA, RNAi) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., RNAi (double-stranded "interfering" RNA), ribonucleoproteins (e.g., iRNPs). The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692- 8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156.
"Oligonucleotide" can include either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
Transcriptional and translational control sequences
The invention provides nucleic acid (e.g., DNA, iRNA) sequences of the invention operatively linked to expression (e.g., transcriptional or translational) control sequence(s), e.g., promoters or enhancers, to direct or modulate RNA synthesis/ expression. The expression control sequence can be in an expression vector. Exemplary bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplary eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I. A promoter sequence can be "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below. As used herein, the term "promoter" includes all sequences capable of driving transcription of a coding sequence in a cell, e.g., a plant cell. Thus, promoters used in the constructs of the invention include crs-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a c/s-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription. "Constitutive" promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation. "Inducible" or "regulatable" promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light. A promoter sequence can be "operably linked to" a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.
"Tissue-specific" promoters are transcriptional control elements that are only active in particular cells or tissues or organs, e.g., in plants or animals. Tissue-specific regulation may be achieved by certain intrinsic factors which ensure that genes encoding proteins specific to a given tissue are expressed. Such factors are known to exist in mammals and plants so as to allow for specific tissues to develop. The term "plant" includes whole plants, plant parts (e.g., leaves, stems, flowers, roots, etc.), plant protoplasts, seeds and plant cells and progeny of same. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous states. As used herein, the term "transgenic plant" includes plants or plant cells into which a heterologous nucleic acid sequence has been inserted, e.g., the nucleic acids and various recombinant constructs (e.g., expression cassettes) of the invention.
Promoters suitable for expressing a polypeptide in bacteria include the E. coli lac or trp promoters, the lad promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter. Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late SV40 promoter, LTRs from retroviruses, and the mouse metallothionein-I promoter. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses may also be used.
Tissue-Specific Plant Promoters
The invention provides expression cassettes that can be expressed in a tissue- specific manner, e.g., that can express an enzyme of the invention in a tissue-specific manner. The invention also provides plants or seeds that express an enzyme of the invention in a tissue-specific manner. The tissue-specificity can be seed specific, stem specific, leaf specific, root specific, fruit specific and the like.
In one aspect, a constitutive promoter such as the CaMV 35S promoter can be used for expression in specific parts of the plant or seed or throughout the plant. For example, for overexpression of an enzyme of the invention, a plant promoter fragment can be employed which will direct expression of a nucleic acid in some or all tissues of a plant, e.g., a regenerated plant. Such "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or T- promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes known to those of skill. Such genes include, e.g., ACTIl from Arabidopsis (Huang (1996) Plant MoI. Biol. 33: 125-139); Caβ from Arabidopsis (GenBank No. U43147, Zhong (1996) MoI. Gen. Genet. 251:196-203); the gene encoding stearoyl-acyl carrier protein desaturase from Brassica napus (Genbank No. X74782, Solocombe ( 1994) Plant Physiol. 104: 1 167-1176); GPcI from maize (GenBank No. Xl 5596; Martinez (1989) J. MoI. Biol 208:551-565); the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant MoI. Biol. 33:97-112); plant promoters described in U.S. Patent Nos. 4,962,028;
5,633,440. The invention uses tissue-specific or constitutive promoters derived from viruses which can include, e.g., the tobamo virus subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92: 1679-1683; the rice tungro bacilliform virus (RTBV), which replicates only in phloem cells in infected rice plants, with its promoter which drives strong phloem-specific reporter gene expression; the cassava vein mosaic virus (CVMV) promoter, with highest activity in vascular elements, in leaf mesophyll cells, and in root tips (Verdaguer (1996) Plant MoI. Biol. 31: 1129-1139).
Alternatively, the plant promoter may direct expression of an enzyme-expressing nucleic acid in a specific tissue, organ or cell type (i.e. tissue-specific promoters) or may be otherwise under more precise environmental or developmental control or under the control of an inducible promoter. Examples of environmental conditions that may affect transcription include anaerobic conditions, elevated temperature, the presence of light, or sprayed with chemicals/hormones. For example, the invention incorporates the drought- inducible promoter of maize (Busk (1997) supra); the cold, drought, and high salt inducible promoter from potato (Kirch (1997) Plant MoI. Biol. 33:897 909).
Tissue-specific promoters can promote transcription only within a certain time frame of developmental stage within that tissue. See, e.g., Blazquez (1998) Plant Cell 10:791-800, characterizing the Arabidopsis LEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77, describing the transcription factor SPL3, which recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene API; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristem promoter eIF4. Tissue specific promoters which are active throughout the life cycle of a particular tissue can be used. In one aspect, the nucleic acids of the invention are operably linked to a promoter active primarily only in cotton fiber cells. In one aspect, the nucleic acids of the invention are operably linked to a promoter active primarily during the stages of cotton fiber cell elongation, e.g., as described by Rinehart (1996) supra. The nucleic acids can be operably linked to the Fbl2A gene promoter to be preferentially expressed in cotton fiber cells (Ibid). See also, John (1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Patent Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promoters and methods for the construction of transgenic cotton plants. Root-specific promoters may also be used to express the nucleic acids of the invention. Examples of root-specific promoters include the promoter from the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol. 123:39-60). Other promoters that can be used to express the nucleic acids of the invention include, e.g., ovule-specific, embryo-specific, endosperm-specific, integument- specific, seed coat-specific promoters, or some combination thereof; a leaf-specific promoter (see, e.g., Busk (1997) Plant J. 11:1285 1295, describing a leaf-specific promoter in maize); the ORF 13 promoter from Agrobacterium rhizogenes (which exhibits high activity in roots, see, e.g., Hansen (1997) supra); a maize pollen specific promoter (see, e.g., Guerrero (1990) MoI. Gen. Genet. 224:161 168); a tomato promoter active during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers can be used (see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specific promoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant MoI. Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa making it a useful tool to target the expression of foreign genes to the epidermal layer of actively growing shoots or fibers; the ovule- specific BELl gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBankNo. U39944); and/or, the promoter in Klee, U.S. Patent No. 5,589,583, describing a plant promoter region is capable of conferring high levels of transcription in meristematic tissue and/or rapidly dividing cells.
Alternatively, plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids of the invention. For example, the invention can use the auxin-response elements El promoter fragment (AuxREs) in the soybean {Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) MoI. Plant Microbe Interact. 10:933-937); and, the promoter responsive to the stress hormone abscisic acid (Sheen (1996) Science 274:1900-1902).
The nucleic acids of the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics. For example, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g. , as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) induced promoters, i.e., promoter responsive to a chemical which can be applied to the transgenic plant in the field, expression of a polypeptide of the invention can be induced at a particular stage of development of the plant. Thus, the invention also provides for transgenic plants containing an inducible gene encoding for polypeptides of the invention whose host range is limited to target plant species, such as corn, rice, barley, wheat, potato or other crops, inducible at any stage of development of the crop.
Tissue-specific plant promoters may drive expression of operably linked sequences in tissues other than the target tissue. Thus, a tissue-specific promoter is one that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well.
The nucleic acids of the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents. These reagents include, e.g., herbicides, synthetic auxins, or antibiotics which can be applied, e.g., sprayed, onto transgenic plants. Inducible expression of the enzyme-producing nucleic acids of the invention will allow the grower to select plants with the optimal starch / sugar ratio. The development of plant parts can thus controlled. In this way the invention provides the means to facilitate the harvesting of plants and plant parts. For example, in various embodiments, the maize In2-2 promoter, activated by benzenesulfonamide herbicide safeners, is used (De Veylder ( 1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. Coding sequences of the invention are also under the control of a tetracycline-inducible promoter, e.g., as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange (1997) Plant J. 1 1: 1315-1324).
If proper polypeptide expression is desired, a polyadenylation region at the 3 '-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from genes in the Agrobacterial T-DNA.
Expression vectors and cloning vehicles
The invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the enzymes and antibodies of the invention. Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), Pl -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as
Bacillus, Aspergillus and yeast). Vectors of the invention can include chromosomal, non- chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Exemplary vectors are include: bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda- ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia);
Eukaryotic: pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector may be used so long as they are replicable and viable in the host. Low copy number or high copy number vectors may be employed with the present invention. In one aspect, the term "expression cassette" comprises a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as an enzyme of the invention) in a host compatible with such sequences. Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers. "Operably linked" as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. Thus, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like. A "vector" comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Patent No. 5,217,879), and includes both the expression and non-expression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector" this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome. The expression vector may comprise a promoter, a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. Mammalian expression vectors can comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences. In some aspects, DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
In one aspect, the expression vectors contain one or more selectable marker genes to permit selection of host cells containing the vector. Such selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for eukaryotic cell culture, genes conferring tetracycline or ampicillin resistance in E. coli, and the S. cerevisiae TRPl gene. Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers. Vectors for expressing the polypeptide or fragment thereof in eukaryotic cells may also contain enhancers to increase expression levels. Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers.
A DNA sequence may be inserted into a vector by a variety of procedures. In general, the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases. Alternatively, blunt ends in both the insert and the vector may be ligated. A variety of cloning techniques are known in the art, e.g., as described in Ausubel and Sambrook.
Such procedures and others are deemed to be within the scope of those skilled in the art.
The vector may be in the form of a plasmid, a viral particle, or a phage. Other vectors include chromosomal, non-chromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by, e.g., Sambrook.
Particular bacterial vectors which may be used include the commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEMl (Promega Biotec, Madison, WI, USA) pQE70, pQE60, pQE-9 (Qiagen), pDIO, psiX174 pBluescript II KS, pNH8A, pNHlόa, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233- 3, DR540, pRIT5 (Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any other vector may be used as long as it is replicable and viable in the host cell.
The nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses and transiently or stably expressed in plant cells and seeds. One exemplary transient expression system uses episomal expression systems, e.g., cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus by transcription of an episomal mini-chromosome containing supercoiled DNA, see, e.g., Covey (1990) Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, coding sequences, i.e., all or sub-fragments of sequences of the invention can be inserted into a plant host cell genome becoming an integral part of the host chromosomal DNA. Sense or antisense transcripts can be expressed in this manner. A vector comprising the sequences (e.g., promoters or coding regions) from nucleic acids of the invention can comprise a marker gene that confers a selectable phenotype on a plant cell or a seed. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or Basta.
Expression vectors capable of expressing nucleic acids and proteins in plants are well known in the art, and can include, e.g., vectors from Agrohacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J. 16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene 173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology 169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology 234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993) Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g., Cecchini (1997) MoI. Plant Microbe Interact. 10:1094-1101), maize Ac/Ds transposable element (see, e.g., Rubin (1997) MoI. Cell. Biol. 17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194), and the maize suppressor-mutator (Spm) transposable element (see, e.g., Schlappi (1996) Plant MoI. Biol. 32:717-725); and derivatives thereof.
In one aspect, the expression vector can have two replication systems to allow it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector can contain at least one sequence homologous to the host cell genome. It can contain two homologous sequences which flank the expression construct. The integrating vector can be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
Expression vectors of the invention may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed, e.g., genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers can also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
Host cells and transformed cells
The invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding an enzyme or an antibody of the invention, or a vector of the invention. The host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Enzymes of the invention can be expressed in any host cell, e.g., any bacterial cell, any yeast cell, e.g., Pichia pastoris, Saccharomyces cerevisiae or Schizosaccharomyces pombe. Exemplary bacterial cells include E. coli, Lactococcus lactis, Streptomyces , Bacillus subtilis, Bacillus cereus, Salmonella typhimurium or any species within the genera Bacillus, Streptomyces and Staphylococcus. Exemplary insect cells include Drosophila S 2 and Spodoptera Sf? . Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art. Techniques for transforming a wide variety of higher plant species are well known and described in the technical and scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477, U.S. Patent No. 5,750,870.
The vector may be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
Where appropriate, the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.
In one aspect, the nucleic acids or vectors of the invention are introduced into the cells for screening, thus, the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO4 precipitation, liposome fusion, lipofection (e.g., LIPOFECTIN™), electroporation, viral infection, etc. The candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction) or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). As many pharmaceutically important screens require human or model mammalian cell targets, retroviral vectors capable of transfecting such targets are preferred.
Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art. The expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps. Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines. The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Depending upon the host employed in a recombinant production procedure, the polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated. Polypeptides of the invention may or may not also include an initial methionine amino acid residue. Cell-free translation systems can also be employed to produce a polypeptide of the invention. Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof. In some aspects, the DNA construct may be linearized prior to conducting an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.
The expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
Amplification of Nucleic Acids
In practicing the invention, nucleic acids encoding the polypeptides of the invention, or modified nucleic acids, can be reproduced by, e.g., amplification. The invention provides amplification primer sequence pairs for amplifying nucleic acids encoding an enzyme, where the primer pairs are capable of amplifying nucleic acids of the invention, including the exemplary SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO.13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO.25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO.33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO.53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID
NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO.87, SEQ ID NO.89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO.105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO.143, SEQ ID NO: 145, SEQ ID NO:147, SEQ ID NO.149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO.159, SEQ ID NO:161, SEQ ID NO.163, SEQ ID NO.165, SEQ ID NO: 167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO.179, SEQ ID NO:181, SEQ ID NO.183, SEQ ID NO:185, SEQ ID NO: 187, SEQ ID NO:189, SEQ ID NO:191 and SEQ ID NO:193. One of skill in the art can design amplification primer sequence pairs for any part of or the full length of these sequences.
Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an array or a blot), detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample. In one aspect of the invention, message isolated from a cell or a cDNA library are amplified. The skilled artisan can select and design suitable oligonucleotide amplification primers. Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241 : 1077; Barringer (1990) Gene 89: 117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86: 1173); and, self-sustained sequence replication (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicase amplification (see, e.g., Smith (1997) J. Clin. Microbiol. 35: 1477-1491), automated Q- beta replicase amplification assay (see, e.g., Burg (1996) MoI. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel;
U.S. Patent Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-564.
The invention also provides amplification primer pairs comprising sequences of the invention, for example, wherein the primer pair comprises a first member having a sequence as set forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more residues of a nucleic acid of the invention, and a second member having a sequence as set forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more residues of the complementary strand of the first member. Determining the degree of sequence identity
The invention provides nucleic acids having various sequence identities (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to exemplary nucleic acids of the invention, including having complete (100%) sequence identity to a nucleic acid of the invention, e.g., an exemplary nucleic acid of the invention (e.g., having a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, etc., all the odd-numbered SEQ ID NO:s through SEQ ID NO:193); and polypeptides having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to a polypeptide of the invention, e.g., an exemplary polypeptide having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10,
SEQ ID NO: 12, etc. In alternative aspects, the sequence identity can be over a region of at least about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive residues, or the full length of the nucleic acid or polypeptide. The extent of sequence identity (homology) may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters.
The phrase "substantially identical" in the context of two nucleic acids or polypeptides, can refer to two or more sequences that have, e.g., at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, nucleotide or amino acid residue (sequence) identity, when compared and aligned for maximum correspondence, as measured using one any known sequence comparison algorithm, as discussed in detail below, or by visual inspection. In alternative aspects, the invention provides nucleic acid and polypeptide sequences having substantial identity to a nucleic acid of the invention, e.g., an exemplary sequence of the invention, over a region of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 residues, or a region ranging from between about 50 residues to the full length of the nucleic acid or polypeptide. Nucleic acid sequences of the invention can be substantially identical over the entire length of a polypeptide coding region.
Additionally a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine). One or more amino acids can be deleted, for example, from an enzyme, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for enzyme activity can be removed.
Homologous sequences also include RNA sequences in which uridines replace the thymines in the nucleic acid sequences. The homologous sequences may be obtained using any of the procedures described herein or may result from the correction of a sequencing error. It will be appreciated that the nucleic acid sequences as set forth herein can be represented in the traditional single character format (see, e.g., Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) or in any other format which records the identity of the nucleotides in a sequence.
Various sequence comparison programs identified herein are used in this aspect of the invention. Protein and/or nucleic acid sequence identities (homologies) may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. MoI. Biol. 215(3):403- 410, 1990; Thompson et al, Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. MoI. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).
Homology or identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of
Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications. The terms "homology" and "identity" in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection. For sequence comparison, one sequence can act as a reference sequence (e.g., an exemplary nucleic acid or polypeptide sequence of the invention) to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A "comparison window", as used herein, includes reference to a segment of any one of the numbers of contiguous residues. For example, in alternative aspects of the invention, continugous residues ranging anywhere from 20 to the full length of an exemplary polypeptide or nucleic acid sequence of the invention, are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. If the reference sequence has the requisite sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, e.g., in alternative aspects, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, that sequence is within the scope of the invention. In alternative embodiments, subsequences ranging from about 20 to 600, about 50 to 200, and about 100 to 150 are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequence for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443, 1970, by the search for similarity method of person & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection. Other algorithms for determining homology or identity include, for example, in addition to a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local Content Program), MACAW (Multiple Alignment Construction & Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences. A number of genome databases are available, for example, a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium (Fraser et al., 1995), M. jannaschii (BuIt et al., 1996), H. influenzae (Fleischmann et al., 1995), E. coli
(Blattner et al., 1997), and yeast (S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al., 2000). Significant progress has also been made in sequencing the genomes of model organism, such as mouse, C. elegans, and Arabadopsis sp. Databases containing genomic information annotated with some functional information are maintained by different organization, and are accessible via the internet.
BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are described, e.g., in Altschul (1977) Nuc. Acids Res. 25:3389-3402; Altschul (1990) J. MoI. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1990) supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N= -4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a references sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. In one aspect, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST"). For example, five specific BLAST programs can be used to perform the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six- frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and, (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256: 1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).
In one aspect of the invention, to determine if a nucleic acid has the requisite sequence identity to be within the scope of the invention, the NCBI BLAST 2.2.2 programs is used, default options to blastp. There are about 38 setting options in the BLAST 2.2.2 program. In this exemplary aspect of the invention, all default values are used except for the default filtering setting (i.e., all parameters set to default except filtering which is set to OFF); in its place a "-F F" setting is used, which disables filtering. Use of default filtering often results in Karlin-Altschul violations due to short length of sequence.
The default values used in this exemplary aspect of the invention include: "Filter for low complexity: ON
Word Size: 3
Matrix: Blosum62
Gap Costs: Existence: 1 1
Extension: 1 " Other default settings are: filter for low complexity OFF, word size of 3 for protein, BLOSUM62 matrix, gap existence penalty of -11 and a gap extension penalty of - 1. An exemplary NCBI BLAST 2.2.2 program setting is set forth in Example 1 , below. Note that the "-W" option defaults to 0. This means that, if not set, the word size defaults to 3 for proteins and 11 for nucleotides.
Computer systems and computer program products
To determine and identify sequence identities, structural homologies, motifs and the like in silico, the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention. As used herein, the words "recorded" and "stored" refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention. As used herein, the terms "computer," "computer program" and "processor" are used in their broadest general contexts and incorporate all such devices, as described in detail, below.
Another aspect of the invention is a computer readable medium having recorded thereon at least one nucleic acid and/or polypeptide sequence of the invention. Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
Aspects of the invention include systems (e.g., internet based systems), particularly computer systems, which store and manipulate the sequences and sequence information described herein. One example of a computer system 100 is illustrated in block diagram form in Figure 1. As used herein, "a computer system" refers to the hardware components, software components, and data storage components used to analyze a nucleotide or polypeptide sequence of the invention. The computer system 100 can include a processor for processing, accessing and manipulating the sequence data. The processor 105 can be any well-known type of central processing unit, such as, for example, the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq, AMD or International Business Machines. The computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.
In one aspect, the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon. The computer system 100 can further include one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110. The data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or a modem capable of connection to a remote data storage system (e.g., via the internet) etc. In some embodiments, the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device. The computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100. Software for accessing and processing the nucleotide or amino acid sequences of the invention can reside in main memory 115 during execution. In some aspects, the computer system 100 may further comprise a sequence comparison algorithm for comparing a nucleic acid sequence of the invention. The algorithm and sequence(s) can be stored on a computer readable medium. A "sequence comparison algorithm" refers to one or more programs which are implemented (locally or remotely) on the computer system 100 to compare a nucleotide sequence with other nucleotide sequences and/or compounds stored within a data storage means. For example, the sequence comparison algorithm may compare the nucleotide sequences of the invention stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies or structural motifs.
The parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied. In some aspects, the parameters may be the default parameters used by the algorithms in the absence of instructions from the user. Figure 2 is a flow diagram illustrating one aspect of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database. The database of sequences can be a private database stored within the computer system 100, or a public database such as GENBANK that is available through the Internet. The process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100. As discussed above, the memory could be any type of memory, including RAM or an internal storage device. The process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison. The process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer. A comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database.
Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system. Once a comparison of the two sequences has been performed at the state 210, a determination is made at a decision state 210 whether the two sequences are the same. Of course, the term "same" is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as "same" in the process 200. If a determination is made that the two sequences are the same, the process 200 moves to a state 214 wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered. Once the name of the stored sequence is displayed to the user, the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database. It should be noted that if a determination had been made at the decision state 212 that the sequences were not homologous, then the process 200 would move immediately to the decision state 218 in order to determine if any other sequences were available in the database for comparison. Accordingly, one aspect of the invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid sequence of the invention and a sequence comparer for conducting the comparison. The sequence comparer may indicate a homology level between the sequences compared or identify structural motifs, or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes. Figure 3 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous. The process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory. The second sequence to be compared is then stored to a memory at a state 256. The process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character of the second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U. If the sequence is a protein sequence, then it can be a single letter amino acid code so that the first and sequence sequences can be easily compared. A determination is then made at a decision state 264 whether the two characters are the same. If they are the same, then the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read. If there are not any more characters to read, then the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user. The level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with an every character in a second sequence, the homology level would be 100%.
Alternatively, the computer program can compare a reference sequence to a sequence of the invention to determine whether the sequences differ at one or more positions. The program can record the length and identity of inserted, deleted or substituted nucleotides or amino acid residues with respect to the sequence of either the reference or the invention. The computer program may be a program which determines whether a reference sequence contains a single nucleotide polymorphism (SNP) with respect to a sequence of the invention, or, whether a sequence of the invention comprises a SNP of a known sequence. Thus, in some aspects, the computer program is a program which identifies SNPs. The method may be implemented by the computer systems described above and the method illustrated in Figure 3. The method can be performed by reading a sequence of the invention and the reference sequences through the use of the computer program and identifying differences with the computer program.
In other aspects the computer based system comprises an identifier for identifying features within a nucleic acid or polypeptide of the invention. An "identifier" refers to one or more programs which identifies certain features within a nucleic acid sequence. For example, an identifier may comprise a program which identifies an open reading frame (ORF) in a nucleic acid sequence. Figure 4 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence. The process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100. The process 300 then moves to a state 306 wherein a database of sequence features is opened. Such a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be "Initiation Codon" and the attribute would be "ATG". Another example would be the feature name "TAATAA Box" and the feature attribute would be "TAATAA". An example of such a database is produced by the University of Wisconsin Genetics Computer Group. Alternatively, the features may be structural polypeptide motifs such as alpha helices, beta sheets, or functional polypeptide motifs such as enzymatic active sites, helix-turn- helix motifs or other motifs known to those skilled in the art. Once the database of features is opened at the state 306, the process 300 moves to a state 308 wherein the first feature is read from the database. A comparison of the attribute of the first feature with the first sequence is then made at a state 310. A determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein the name of the found feature is displayed to the user. The process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324. However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute of the next feature is compared against the first sequence. If the feature attribute is not found in the first sequence at the decision state 316, the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database. Thus, in one aspect, the invention provides a computer program that identifies open reading frames (ORFs).
A polypeptide or nucleic acid sequence of the invention may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, a sequence can be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many computer programs and databases may be used as sequence comparison algorithms, identifiers, or sources of reference nucleotide sequences or polypeptide sequences to be compared to a nucleic acid sequence of the invention. The programs and databases used to practice the invention include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. MoI. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius2.DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations
Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwent's World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure. Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
Hybridization of nucleic acids
The invention provides isolated, synthetic or recombinant nucleic acids that hybridize under various stringent conditions to a nucleic acid of the invention, e.g., an exemplary sequence of the invention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, etc., and subsequences thereof (which include complementary sequences), or a nucleic acid that encodes a polypeptide of the invention. The stringent conditions can be highly stringent conditions, medium stringent conditions, low stringent conditions, including the high and reduced stringency conditions described herein.
"Hybridization" refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. For example, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below. In alternative aspects, nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low), as set forth herein.
In alternative embodiments, nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of nucleic acid of the invention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues in length. Nucleic acids shorter than full length are also included. These nucleic acids can be useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA, antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites and the like. In one aspect, nucleic acids of the invention are defined by their ability to hybridize under high stringency comprises conditions of about 50% formamide at about 37°C to 42°C. In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 3O0C to 35°C.
Alternatively, nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 420C in 50% formamide, 5X SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA). In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 350C.
Following hybridization, the filter may be washed with 6X SSC, 0.5% SDS at 500C. These conditions are considered to be "moderate" conditions above 25% formamide and "low" conditions below 25% formamide. A specific example of
"moderate" hybridization conditions is when the above hybridization is conducted at 30% formamide. A specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 10% formamide.
The temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and low stringency conditions as set forth in Ausubel and Sambrook. Variations on the above ranges and conditions are well known in the art. Hybridization conditions are discussed further, below. The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence. For example, to obtain nucleic acids of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 50C from 680C to 42°C in a hybridization buffer having a Na+ concentration of approximately IM. Following hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate" conditions above 500C and "low" conditions below 500C. A specific example of "moderate" hybridization conditions is when the above hybridization is conducted at 55°C A specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 45°C.
Alternatively, the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C. In this case, the concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe. Following hybridization, the filter may be washed with 6X SSC, 0.5% SDS at 500C. These conditions are considered to be "moderate" conditions above 25% formamide and "low" conditions below 25% formamide. A specific example of "moderate" hybridization conditions is when the above hybridization is conducted at 30% formamide. A specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 10% formamide.
However, the selection of a hybridization format is not critical - it is the stringency of the wash conditions that set forth the conditions which determine whether a nucleic acid is within the scope of the invention. Wash conditions used to identify nucleic acids within the scope of the invention include, e.g.: a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 500C or about 55°C to about 600C; or, a salt concentration of about 0.15 M NaCl at 72°C for about 15 minutes; or, a salt concentration of about 0.2X SSC at a temperature of at least about 500C or about 550C to about 600C for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1X SSC containing 0.1% SDS at 68oC for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen and Ausubel for a description of SSC buffer and equivalent conditions. These methods may be used to isolate nucleic acids of the invention.
Oligonucleotides probes and methods for using them
The invention also provides nucleic acid probes for identifying nucleic acids encoding a polypeptide with an enzyme activity. In one aspect, the probe comprises at least 10 consecutive bases of a nucleic acid of the invention. Alternatively, a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 150, 160, 170, 180, 190, 200 or more, or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a nucleic acid of the invention. The probes identify a nucleic acid by binding and/or hybridization. The probes can be used in arrays of the invention, see discussion below, including, e.g., capillary arrays. The probes of the invention can also be used to isolate other nucleic acids or polypeptides.
The probes of the invention can be used to determine whether a biological sample, such as a soil sample, contains an organism having a nucleic acid sequence of the invention (e.g., an enzyme-encoding nucleic acid) or an organism from which the nucleic acid was obtained. In such procedures, a biological sample potentially harboring the organism from which the nucleic acid was isolated is obtained and nucleic acids are obtained from the sample. The nucleic acids are contacted with the probe under conditions which permit the probe to specifically hybridize to any complementary sequences present in the sample. Where necessary, conditions which permit the probe to specifically hybridize to complementary sequences may be determined by placing the probe in contact with complementary sequences from samples known to contain the complementary sequence, as well as control sequences which do not contain the complementary sequence. Hybridization conditions, such as the salt concentration of the hybridization buffer, the formamide concentration of the hybridization buffer, or the hybridization temperature, may be varied to identify conditions which allow the probe to hybridize specifically to complementary nucleic acids (see discussion on specific hybridization conditions).
If the sample contains the organism from which the nucleic acid was isolated, specific hybridization of the probe is then detected. Hybridization may be detected by labeling the probe with a detectable agent such as a radioactive isotope, a fluorescent dye or an enzyme capable of catalyzing the formation of a detectable product. Many methods for using the labeled probes to detect the presence of complementary nucleic acids in a sample are familiar to those skilled in the art. These include Southern Blots, Northern Blots, colony hybridization procedures, and dot blots. Protocols for each of these procedures are provided in Ausubel and Sambrook.
Alternatively, more than one probe (at least one of which is capable of specifically hybridizing to any complementary sequences which are present in the nucleic acid sample), may be used in an amplification reaction to determine whether the sample contains an organism containing a nucleic acid sequence of the invention (e.g., an organism from which the nucleic acid was isolated). In one aspect, the probes comprise oligonucleotides. In one aspect, the amplification reaction may comprise a PCR reaction. PCR protocols are described in Ausubel and Sambrook (see discussion on amplification reactions). In such procedures, the nucleic acids in the sample are contacted with the probes, the amplification reaction is performed, and any resulting amplification product is detected. The amplification product may be detected by performing gel electrophoresis on the reaction products and staining the gel with an intercalator such as ethidium bromide. Alternatively, one or more of the probes may be labeled with a radioactive isotope and the presence of a radioactive amplification product may be detected by autoradiography after gel electrophoresis.
Probes derived from sequences near the 3' or 5' ends of a nucleic acid sequence of the invention can also be used in chromosome walking procedures to identify clones containing additional, e.g., genomic sequences. Such methods allow the isolation of genes which encode additional proteins of interest from the host organism.
In one aspect, nucleic acid sequences of the invention are used as probes to identify and isolate related nucleic acids. In some aspects, the so-identified related nucleic acids may be cDNAs or genomic DNAs from organisms other than the one from which the nucleic acid of the invention was first isolated. In such procedures, a nucleic acid sample is contacted with the probe under conditions which permit the probe to specifically hybridize to related sequences. Hybridization of the probe to nucleic acids from the related organism is then detected using any of the methods described above.
In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Hybridization may be carried out under conditions of low stringency, moderate stringency or high stringency. As an example of nucleic acid hybridization, a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45°C in a solution consisting of 0.9 M NaCl, 50 mM NaH2PO4, pH 7.0, 5.0 mM Na2EDTA, 0.5% SDS, 1OX Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2 X 107 cpm (specific activity 4-9 X 108 cpm/ug) of 32P end-labeled oligonucleotide probe are then added to the solution. After 12-16 hours of incubation, the membrane is washed for 30 minutes at room temperature (RT) in IX SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh IX SET at Tm-IO0C for the oligonucleotide probe. The membrane is then exposed to autoradiographic film for detection of hybridization signals.
By varying the stringency of the hybridization conditions used to identify nucleic acids, such as cDNAs or genomic DNAs, which hybridize to the detectable probe, nucleic acids having different levels of homology to the probe can be identified and isolated.
Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes. The melting temperature, Tm, is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly complementary probe. Very stringent conditions are selected to be equal to or about 5°C lower than the Tm for a particular probe. The melting temperature of the probe may be calculated using the following exemplary formulas. For probes between 14 and 70 nucleotides in length the melting temperature (Tm) is calculated using the formula: Tm=81.5+16.6(log [Na+])+0.41 (fraction G+C)-(600/N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting temperature may be calculated using the equation: Tm=81.5+16.6(log
[Na+])+0.41 (fraction G+C)-(0.63% formamide)-(600/N) where N is the length of the probe. Prehybridization may be carried out in 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOOμg denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, lOOμg denatured fragmented salmon sperm DNA, 50% formamide. Formulas for SSC and Denhardt's and other solutions are listed, e.g., in Sambrook.
In one aspect, hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-250C below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 5-100C below the Tm. In one aspect, hybridizations in 6X SSC are conducted at approximately 68°C. In one aspect, hybridizations in 50% formamide containing solutions are conducted at approximately 42°C. All of the foregoing hybridizations would be considered to be under conditions of high stringency.
In one aspect, following hybridization, the filter is washed to remove any non- specifically bound detectable probe. The stringency used to wash the filters can also be varied depending on the nature of the nucleic acids being hybridized, the length of the nucleic acids being hybridized, the degree of complementarity, the nucleotide sequence composition (e.g., GC v. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examples of progressively higher stringency condition washes are as follows: 2X SSC, 0.1 % SDS at room temperature for 15 minutes (low stringency); 0. IX SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0. IX SSC, 0.5% SDS for 15 to 30 minutes at between the hybridization temperature and 68°C (high stringency); and 0.15M NaCl for 15 minutes at 72°C (very high stringency). A final low stringency wash can be conducted in 0. IX SSC at room temperature. The examples above are merely illustrative of one set of conditions that can be used to wash filters. One of skill in the art would know that there are numerous recipes for different stringency washes.
Nucleic acids which have hybridized to the probe can be identified by autoradiography or other conventional techniques. The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence.
For example, to obtain nucleic acids of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 5°C from 680C to 42°C in a hybridization buffer having a Na+ concentration of approximately IM. Following hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be "moderate" conditions above 500C and "low" conditions below 500C. An example of "moderate" hybridization conditions is when the above hybridization is conducted at 55°C. An example of "low stringency" hybridization conditions is when the above hybridization is conducted at 45°C. Alternatively, the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C. In this case, the concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe. Following hybridization, the filter may be washed with 6X SSC, 0.5% SDS at 500C. These conditions are considered to be "moderate" conditions above 25% formamide and "low" conditions below 25% formamide. A specific example of "moderate" hybridization conditions is when the above hybridization is conducted at 30% formamide. A specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 10% formamide. These probes and methods of the invention can be used to isolate, or identify (e.g., using an array), nucleic acids having a sequence with at least about 950%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity to a nucleic acid sequence of the invention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive bases thereof, and the sequences complementary thereto. Homology may be measured using an alignment algorithm, as discussed herein. For example, the homologous polynucleotides may have a coding sequence which is a naturally occurring allelic variant of one of the coding sequences described herein. Such allelic variants may have a substitution, deletion or addition of one or more nucleotides when compared to a nucleic acid of the invention. Additionally, the probes and methods of the invention may be used to isolate, or identify (e.g., using an array), nucleic acids which encode polypeptides having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity (homology) to a polypeptide of the invention comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutive amino acids thereof as determined using a sequence alignment algorithm, e.g., such as the FASTA version 3.0t78 algorithm with the default parameters, or a BLAST 2.2.2 program with exemplary settings as set forth herein. Inhibiting Expression of Enzymes
The invention further provides for nucleic acids complementary to (e.g., antisense sequences to) the nucleic acid sequences of the invention, e.g., enzyme-encoding sequences. Antisense sequences are capable of inhibiting the transport, splicing or transcription of enzyme-encoding genes. The inhibition can be effected through the targeting of genomic DNA or messenger RNA. The inhibition can be effected using DNA, e.g., an inhibitory ribozyme, or an RNA, e.g., a double-stranded iRNA (e.g., siRNA, miRNA), comprising a sequence of the invention. The transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage.
The invention provides a set of inhibitors comprising oligonucleotides capable of binding enzyme gene and/or message, in either case preventing or inhibiting the production or function of enzyme. The association can be through sequence specific hybridization. Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of enzyme message. The oligonucleotide can have enzyme activity which causes such cleavage, such as ribozymes. The oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. One may screen a pool of many different such oligonucleotides for those with the desired activity.
Antisense Oligonucleotides The invention provides antisense oligonucleotides capable of binding enzyme message which can inhibit enzyme activity by targeting mRNA or genomic DNA. Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such enzyme oligonucleotides using the novel reagents of the invention. For example, gene walking/ RNA mapping protocols to screen for effective antisense oligonucleotides are well known in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183, describing an RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for potent antisense sequence selection. See also Smith (2000) Eur. J. Pharm. Sci. 11:191-198. In one aspect, recombinantly generated, or, isolated naturally occurring nucleic acids are used as antisense oligonucleotides. The antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The antisense oligonucleotides can be single stranded or double-stranded RNA or DNA. The optimal length can be determined by routine screening. The antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144: 189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N. J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, and morpholino carbamate nucleic acids, as described above.
Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense enzyme sequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
Inhibitory Ribozymes
The invention provides for with ribozymes capable of binding enzyme message that can inhibit enzyme activity by targeting mRNA. Strategies for designing ribozymes and selecting the enzyme-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary basepairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.
In some circumstances, the enzymatic nature of a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide. This potential advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted
RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RnaseP-like RNA (in association with an RNA guide sequence). Examples of such hammerhead motifs are described by Rossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry 31 :16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S. Pat. No. 4,987,071. The recitation of these specific motifs is not intended to be limiting; those skilled in the art will recognize that an enzymatic RNA molecule of this invention has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule. RNA interference (RNAi)
In one aspect, the invention provides an RNA inhibitory molecule, a so-called "RNAi" molecule, comprising an enzyme sequence of the invention. The RNAi, or RNA interference molecules include small interfering RNA, or siRNAs, for inhibiting transcription, and microRNAs, or miRNAs, for inhibiting translation. The RNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The
RNAi can inhibit expression of an enzyme gene. In one aspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length. While the invention is not limited by any particular mechanism of action, the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). A possible basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In one aspect, the RNAi's of the invention are used in gene- silencing therapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7: 1040-1046. In one aspect, the invention provides methods to selectively degrade RNA using the RNAi's of the invention. The process may be practiced in vitro, ex vivo or in vivo. In one aspect, the RNAi molecules of the invention can be used to generate a loss-of- function mutation in a cell, an organ or an animal. Methods for making and using RNAi molecules for selectively degrade KNA are well known in the art, see, e.g., U.S. Patent No. 6,506,559; 6,511,824; 6,515,109; 6,489,127.
Modification of Nucleic Acids The invention provides methods of generating variants of the nucleic acids and polypeptides of the invention, e.g., nucleic acids encoding an enzyme, peptide or an antibody of the invention. These methods can be repeated or used in various combinations to generate enzymes or antibodies having an altered or different activity or an altered or different stability from that of an enzyme or antibody encoded by the template nucleic acid. These methods also can be repeated or used in various combinations, e.g., to generate variations in gene/ message expression, message translation or message stability. In another aspect, the genetic composition of a cell is altered by, e.g., modification of a homologous gene ex vivo, followed by its reinsertion into the cell. The term "variant" can include polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of an enzyme of the invention. Variants can be produced by any number of means included methods such as, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and any combination thereof. Techniques for producing variant enzymes having activity at a pH or temperature, for example, that is different from a wild-type enzyme, are included herein. The term "saturation mutagenesis", also called Gene Site Saturation Mutagenesis, abbreviated as "GSSM", includes a method that uses degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, as described in detail, below. The term "optimized directed evolution system" or "optimized directed evolution" includes a method for reassembling fragments of related nucleic acid sequences, e.g., related genes, and explained in detail, below. The term "synthetic ligation reassembly" or "SLR" includes a method of ligating oligonucleotide fragments in a non-stochastic fashion, and explained in detail, below.
A nucleic acid of the invention can be altered by any means. For example, random or stochastic methods, or, non-stochastic, or "directed evolution," methods, see, e.g., U.S. Patent No. 6,361,974. Methods for random mutation of genes are well known in the art, see, e.g., U.S. Patent No. 5,830,696. For example, mutagens can be used to randomly mutate a gene. Mutagens include, e.g., ultraviolet light or gamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, to induce DNA breaks amenable to repair by recombination. Other chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other mutagens are analogues of nucleotide precursors, e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. These agents can be added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.
Any technique in molecular biology can be used, e.g., random PCR mutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471; or, combinatorial multiple cassette mutagenesis, see, e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleic acids, e.g., genes, can be reassembled after random, or "stochastic," fragmentation, see, e.g., U.S. Patent Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721 ; 5,824,514; 5,811,238; 5,605,793. In alternative aspects, modifications, additions or deletions are introduced by error-prone PCR, shuffling, oligonucleotide- directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM) synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid mul timer creation, and/or a combination of these and other methods.
The following publications describe a variety of recursive recombination procedures and/or methods which can be incorporated into the methods of the invention: Stemmer (1999) "Molecular breeding of viruses for targeting and other clinical properties" Tumor Targeting 4: 1-4; Ness (1999) Nature Biotechnology 17:893-896; Chang (1999) "Evolution of a cytokine using DNA family shuffling" Nature Biotechnology 17:793-797; Minshull (1999) "Protein evolution by molecular breeding" Current Opinion in Chemical Biology 3:284-290; Christians (1999) "Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling" Nature Biotechnology 17:259-264; Crameri (1998) "DNA shuffling of a family of genes from diverse species accelerates directed evolution" Nature 391:288-291; Crameri (1997) "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature Biotechnology 15:436-438; Zhang (1997) "Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening" Proc. Natl. Acad. Sci. USA 94:4504-4509; Patten et al. (1997) "Applications of DNA Shuffling to Pharmaceuticals and Vaccines" Current Opinion in Biotechnology 8:724-733; Crameri et al. (1996) "Construction and evolution of antibody-phage libraries by DNA shuffling" Nature Medicine 2:100-103; Gates et al. (1996) "Affinity selective isolation of ligands from peptide libraries through display on a lac repressor headpiece dimer " Journal of Molecular Biology 255:373-386; Stemmer (1996) "Sexual PCR and Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH Publishers, New York, pp.447-457; Crameri and Stemmer (1995) "Combinatorial multiple cassette mutagenesis creates all the permutations of mutant and wildtype cassettes" BioTechniques 18:194-195; Stemmer et al. (1995) "Single-step assembly of a gene and entire plasmid form large numbers of oligodeoxyribonucleotides" Gene, 164:49-53; Stemmer (1995) "The Evolution of Molecular Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence Space" Bio/Technology 13:549-553; Stemmer (1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature 370:389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution." Proc. Natl. Acad. Sci. USA 91:10747-10751.
Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview" Anal Biochem. 254(2): 157-178; Dale et al. (1996) "Oligonucleotide-directed random mutagenesis using the phosphorothioate method" Methods MoI. Biol. 57:369-374; Smith (1985) "In vitro mutagenesis" Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985) "Strategies and applications of in vitro mutagenesis" Science 229: 1193-1201; Carter (1986) "Site-directed mutagenesis" Biochem. J. 237: 1-7; and Kunkel (1987) "The efficiency of oligonucleotide directed mutagenesis" in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel (1985) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and efficient site-specific mutagenesis without phenotypic selection" Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant Trp repressors with new DNA-binding specificities" Science 242:240-245); oligonucleotide- directed mutagenesis (Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982) "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith
(1983) "Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors" Methods in Enzymol. 100:468-500; and Zoller & Smith (1987) Oligonucleotide- directed mutagenesis: a simple method using two oligonucleotide primers and a single- stranded DNA template" Methods in Enzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985) "The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764; Taylor et al. (1985) "The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) "Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis" Nucl. Acids Res. 14: 9679-9698; Sayers et al. (1988) "Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl. Acids Res. 16:791- 802; and Sayers et al. (1988) "Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide" Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al.
(1984) "The gapped duplex DNA approach to oligonucleotide-directed mutation construction" Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol. "Oligonucleotide-directed construction of mutations via gapped duplex DNA" 154:350-367; Kramer et al. (1988) "Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations" Nucl. Acids Res. 16: 7207; and Fritz et al. (1988) "Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999).
Additional protocols used in the methods of the invention include point mismatch repair (Kramer (1984) "Point Mismatch Repair" Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter et al. (1985) "Improved oligonucleotide site-directed mutagenesis using M13 vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using Ml 3 vectors" Methods in Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) "Use of oligonucleotides to generate large deletions" Nucl. Acids Res. 14: 5115), restriction- selection and restriction-selection and restriction-purification (Wells et al. (1986) "Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984) "Total synthesis and cloning of a gene coding for the ribonuclease S protein" Science 223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide- binding protein (transducin)" Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985) "Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites" Gene 34:315-323; and Grundstrom et al. (1985) "Oligonucleotide-directed mutagenesis by microscale shot-gun' gene synthesis" Nucl. Acids Res. 13: 3305-3316), double-strand break repair (Mandecki (1986); Arnold (1993) "Protein engineering for unusual environments" Current Opinion in Biotechnology 4:450-455. "Oligonucleotide- directed double-strand break repair in plasmids of Escherichia coli: a method for site- specific mutagenesis" Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.
Additional protocols used in the methods of the invention include those discussed in U.S. Patent Nos. 5,605,793 to Stemmer (Feb. 25, 1997), "Methods for In Vitro Recombination;" U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep. 22, 1998) "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), "DNA
Mutagenesis by Random Fragmentation and Reassembly;" U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) "End-Complementary Polymerase Reaction;" U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), "Methods and Compositions for Cellular and Metabolic Engineering;" WO 95/22625, Stemmer and Crameri, "Mutagenesis by Random Fragmentation and Reassembly;" WO 96/33207 by Stemmer and Lipschutz "End Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;" WO 97/35966 by Minshull and Stemmer, "Methods and Compositions for Cellular and Metabolic Engineering;" WO 99/41402 by Punnonen et al. "Targeting of Genetic Vaccine Vectors;" WO 99/41383 by Punnonen et al. "Antigen Library Immunization;" WO 99/41369 by Punnonen et al. "Genetic Vaccine Vector Engineering;" WO 99/41368 by Punnonen et al. "Optimization of Immunomodulatory Properties of Genetic Vaccines;" EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation and Reassembly;" EP 0932670 by Stemmer "Evolving Cellular DNA Uptake by Recursive Sequence Recombination;" WO 99/23107 by Stemmer et al., "Modification of Virus Tropism and Host Range by Viral Genome Shuffling;" WO 99/21979 by Apt et al., "Human Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al. "Evolution of Whole Cells and Organisms by Recursive Sequence Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering;" WO 98/27230 by Stemmer et al., "Methods for Optimization of Gene Therapy by Recursive Sequence Shuffling and Selection," WO 00/00632, "Methods for Generating Highly Diverse Libraries," WO 00/09679, "Methods for Obtaining in Vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences," WO 98/42832 by Arnold et al., "Recombination of
Polynucleotide Sequences Using Random or Defined Primers," WO 99/29902 by Arnold et al., "Method for Creating Polynucleotide and Polypeptide Sequences," WO 98/41653 by Vind, "An in Vitro Method for Construction of a DNA Library," WO 98/41622 by Borchert et al., "Method for Constructing a Library Using DNA Shuffling," and WO 98/42727 by Pati and Zarling, "Sequence Alterations using Homologous Recombination."
Protocols that can be used to practice the invention (providing details regarding various diversity generating methods) are described, e.g., in U.S. Patent application serial no. (USSN) 09/407,800, "SHUFFLING OF CODON ALTERED GENES" by Patten et al. filed Sep. 28, 1999; "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION" by del Cardayre et al., United States Patent No. 6,379,964; "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION" by Crameri et al., United States Patent Nos. 6,319,714; 6,368,861; 6,376,246; 6,423,542; 6,426,224 and PCT/USOO/01203; "USE OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING" by Welch et al., United States Patent No. 6,436,675; "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed Jan. 18, 2000, (PCT/USOO/01202) and, e.g. "METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed JuI. 18, 2000 (U.S. Ser. No. 09/618,579); "METHODS OF POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer, filed Jan. 18, 2000 (PCT/USOO/01138); and "SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION" by Affholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and United States Patent Nos. 6,177,263; 6,153,410.
Non-stochastic, or "directed evolution," methods include, e.g., saturation mutagenesis, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof are used to modify the nucleic acids of the invention to generate enzymes with new or altered properties (e.g., activity under highly acidic or alkaline conditions, high temperatures, and the like). Polypeptides encoded by the modified nucleic acids can be screened for an activity before testing for proteolytic or other activity. Any testing modality or protocol can be used, e.g., using a capillary array platform. See, e.g., U.S. Patent Nos. 6,361,974; 6,280,926; 5,939,250. Saturation mutagenesis, or, GSSM
In one aspect of the invention, non-stochastic gene modification, a "directed evolution process," is used to generate enzymes and antibodies with new or altered properties. Variations of this method have been termed "Gene Site Saturation Mutagenesis™, "site-saturation mutagenesis," "saturation mutagenesis" or simply "GSSM™." It can be used in combination with other mutagenization processes. See, e.g., U.S. Patent Nos. 6,171,820; 6,238,884. Thus, the invention provides methods for making enzyme using Gene Site Saturation mutagenesis, or, GSSM, as described herein, and also in U.S. Patent Nos. 6,171,820; 6,238,884, and 6,579,258.
In one aspect, GSSM comprises providing a template polynucleotide and a plurality of oligonucleotides, wherein each oligonucleotide comprises a sequence homologous to the template polynucleotide, thereby targeting a specific sequence of the template polynucleotide, and a sequence that is a variant of the homologous gene; generating progeny polynucleotides comprising non-stochastic sequence variations by replicating the template polynucleotide with the oligonucleotides, thereby generating polynucleotides comprising homologous gene sequence variations.
In one aspect, codon primers containing a degenerate N,N,G/T sequence are used to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position, e.g., an amino acid residue in an enzyme active site or ligand binding site targeted to be modified. These oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N,N,G/T sequence, and, optionally, a second homologous sequence. The downstream progeny translational products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids. In one aspect, one such degenerate oligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions. In another aspect, at least two degenerate cassettes are used - either in the same oligonucleotide or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions. For example, more than one N,N,G/T sequence can be contained in one oligonucleotide to introduce amino acid mutations at more than one site. This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s). In another aspect, oligonucleotides serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.
In one aspect, simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In another aspect, degenerate cassettes having less degeneracy than the N,N,G/T sequence are used. For example, it may be desirable in some instances to use (e.g. in an oligonucleotide) a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet. Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet. Alternatively, it may be desirable in some instances to use (e.g. in an oligo) a degenerate N,N,N triplet sequence.
In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets) allows for systematic and easy generation of a full range of possible natural amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide (in alternative aspects, the methods also include generation of less than all possible substitutions per amino acid residue, or codon, position). For example, for a 100 amino acid polypeptide, 2000 distinct species (i.e. 20 possible amino acids per position X 100 amino acid positions) can be generated. Through the use of an oligonucleotide or set of oligonucleotides containing a degenerate N,N,G/T triplet, 32 individual sequences can code for all 20 possible natural amino acids. Thus, in a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using at least one such oligonucleotide, there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides. In contrast, the use of a non-degenerate oligonucleotide in site- directed mutagenesis leads to only one progeny polypeptide product per reaction vessel. Nondegenerate oligonucleotides can optionally be used in combination with degenerate primers disclosed; for example, nondegenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide. This provides one means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.
In one aspect, each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 natural amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide (other aspects use less than all 20 natural combinations). The 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g. cloned into a suitable host, e.g., E. coli host, using, e.g., an expression vector) and subjected to expression screening. When an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide, such as increased proteolytic activity under alkaline or acidic conditions), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein.
In one aspect, upon mutagenizing each and every amino acid position in a parental polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes may be identified at more than one amino acid position. One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions. For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Thus, there are 3 x 3 x 3 or 27 total possibilities, including 7 that were previously examined - 6 single point mutations (i.e. 2 at each of three positions) and no change at any position. In another aspect, site-saturation mutagenesis can be used together with another stochastic or non-stochastic means to vary sequence, e.g., synthetic ligation reassembly (see below), shuffling, chimerization, recombination and other mutagenizing processes and mutagenizing agents. This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.
Synthetic Ligation Reassembly (SLR)
The invention provides a non-stochastic gene modification system termed "synthetic ligation reassembly," or simply "SLR," a "directed evolution process," to generate enzymes and antibodies with new or altered properties. SLR is a method of ligating oligonucleotide fragments together non-stochastically. This method differs from stochastic oligonucleotide shuffling in that the nucleic acid building blocks are not shuffled, concatenated or chimerized randomly, but rather are assembled non- stochastically. See, e.g., U.S. Patent Nos. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776. In one aspect, SLR comprises the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucleotide to generate polynucleotides comprising homologous gene sequence variations. SLR does not depend on the presence of high levels of homology between polynucleotides to be rearranged. Thus, this method can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10 different chimeras. SLR can be used to generate libraries comprised of over io1000 different progeny chimeras. Thus, aspects of the present invention include non-stochastic methods of producing a set of finalized chimeric nucleic acid molecule shaving an overall assembly order that is chosen by design. This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved. The mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders. Thus, the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends. If more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s). In one aspect, the annealed building pieces are treated with an enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent bonding of the building pieces. In one aspect, the design of the oligonucleotide building blocks is obtained by analyzing a set of progenitor nucleic acid sequence templates that serve as a basis for producing a progeny set of finalized chimeric polynucleotides. These parental oligonucleotide templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, e.g., chimerized or shuffled. In one aspect of this method, the sequences of a plurality of parental nucleic acid templates are aligned in order to select one or more demarcation points. The demarcation points can be located at an area of homology, and are comprised of one or more nucleotides. These demarcation points are preferably shared by at least two of the progenitor templates. The demarcation points can thereby be used to delineate the boundaries of oligonucleotide building blocks to be generated in order to rearrange the parental polynucleotides. The demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules. A demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences. Alternatively, a demarcation point can be an area of homology that is shared by at least half of the parental polynucleotide sequences, or, it can be an area of homology that is shared by at least two thirds of the parental polynucleotide sequences. Even more preferably a serviceable demarcation points is an area of homology that is shared by at least three fourths of the parental polynucleotide sequences, or, it can be shared by at almost all of the parental polynucleotide sequences. In one aspect, a demarcation point is an area of homology that is shared by all of the parental polynucleotide sequences.
In one aspect, a ligation reassembly process is performed exhaustively in order to generate an exhaustive library of progeny chimeric polynucleotides. In other words, all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules. At the same time, in another aspect, the assembly order (i.e. the order of assembly of each building block in the 5' to 3 sequence of each finalized chimeric nucleic acid) in each combination is by design (or non- stochastic) as described above. Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.
In another aspect, the ligation reassembly method is performed systematically. For example, the method is performed in order to generate a systematically compartmentalized library of progeny molecules, with compartments that can be screened systematically, e.g. one by one. In other words this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, a design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups. Because of its ability to perform chimerizations in a manner that is highly flexible yet exhaustive and systematic as well, particularly when there is a low level of homology among the progenitor molecules, these methods provide for the generation of a library (or set) comprised of a large number of progeny molecules. Because of the non-stochastic nature of the instant ligation reassembly invention, the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design. The saturation mutagenesis and optimized directed evolution methods also can be used to generate different progeny molecular species. It is appreciated that the invention provides freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings. It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobble, i.e. the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding progenitor template. Alternatively, a codon can be altered such that the coding for an originally amino acid is altered. This invention provides that such substitutions can be introduced into the nucleic acid building block in order to increase the incidence of intermolecular homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.
In another aspect, the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g. by mutagenesis) or in an in vivo process (e.g. by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.
In one aspect, a nucleic acid building block is used to introduce an intron. Thus, functional introns are introduced into a man-made gene manufactured according to the methods described herein. The artificially introduced intron(s) can be functional in a host cells for gene splicing much in the way that naturally-occurring introns serve functionally in gene splicing.
Optimized Directed Evolution System
The invention provides a non-stochastic gene modification system termed "optimized directed evolution system" to generate enzymes and antibodies with new or altered properties. Optimized directed evolution is directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of nucleic acids through recombination. Optimized directed evolution allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.
A crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence. This method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
In addition, this method provides a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems. Previously, if one generated, for example, 1013 chimeric molecules during a reaction, it would be extremely difficult to test such a high number of chimeric variants for a particular activity. Moreover, a significant portion of the progeny population would have a very high number of crossover events which resulted in proteins that were less likely to have increased levels of a particular activity. By using these methods, the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events. Thus, although one can still generate 1013 chimeric molecules during a reaction, each of the molecules chosen for further analysis most likely has, for example, only three crossover events. Because the resulting progeny population can be skewed to have a predetermined number of crossover events, the boundaries on the functional variety between the chimeric molecules is reduced. This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait.
One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides corresponding to fragments or portions of each parental sequence. Each oligonucleotide in one aspect includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order. Alternatively protocols for practicing these methods of the invention can be found in U.S. Patent Nos. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.
The number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created. For example, three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature. As one example, a set of 50 oligonucleotide sequences can be generated corresponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences. The probability that each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a 1/3 chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.
Accordingly, a probability density function (PDF) can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides corresponding to each variant, and the concentrations of each variant during each step in the ligation reaction. The statistics and mathematics behind determining the PDF is described below. By utilizing these methods, one can calculate such a probability density function, and thus enrich the chimeric progeny population for a predetermined number of crossover events resulting from a particular ligation reaction. Moreover, a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events. These methods are directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of a nucleic acid encoding a polypeptide through recombination. This system allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events. A crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence. The method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.
Determining Crossover Events
Aspects of the invention include a system and software that receive a desired crossover probability density function (PDF), the number of parent genes to be reassembled, and the number of fragments in the reassembly as inputs. The output of this program is a "fragment PDF" that can be used to determine a recipe for producing reassembled genes, and the estimated crossover PDF of those genes. The processing described herein is preferably performed in MATLAB™ (The Mathworks, Natick, Massachusetts) a programming language and development environment for technical computing.
Iterative Processes
In practicing the invention, these processes can be iteratively repeated. For example a nucleic acid (or, the nucleic acid) responsible for an altered enzyme or antibody phenotype is identified, re-isolated, again modified, re-tested for activity. This process can be iteratively repeated until a desired phenotype is engineered. For example, an entire biochemical anabolic or catabolic pathway can be engineered into a cell, including proteolytic activity. Similarly, if it is determined that a particular oligonucleotide has no affect at all on the desired trait (e.g., a new enzyme phenotype), it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed. Since incorporating the sequence within a larger sequence prevents any crossover events, there will no longer be any variation of this sequence in the progeny polynucleotides. This iterative practice of determining which oligonucleotides are most related to the desired trait, and which are unrelated, allows more efficient exploration all of the possible protein variants that might be provide a particular trait or activity.
In vivo shuffling
In vivo shuffling of molecules is use in methods of the invention that provide variants of polypeptides of the invention, e.g., antibodies, enzymes, and the like. In vivo shuffling can be performed utilizing the natural property of cells to recombine multimers. While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules. The formation of the chiasma requires the recognition of homologous sequences.
In one aspect, the invention provides a method for producing a hybrid polynucleotide from at least a first polynucleotide and a second polynucleotide. The invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell. The regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide. The term "hybrid polynucleotide", as used herein, is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences. Such hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules. In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.
Producing sequence variants
The invention also provides methods of making sequence variants of the nucleic acid and enzyme and antibody sequences of the invention or isolating enzymes using the nucleic acids and polypeptides of the invention. In one aspect, the invention provides for variants of a protein-encoding gene of the invention, which can be altered by any means, including, e.g., random or stochastic methods, or, non-stochastic, or "directed evolution," methods, as described above. The isolated variants may be naturally occurring. Variant can also be created in vitro. Variants may be created using genetic engineering techniques such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives may be created using chemical synthesis or modification procedures. Other methods of making variants are also familiar to those skilled in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids which encode polypeptides having characteristics which enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. These nucleotide differences can result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
For example, variants may be created using error prone PCR. In error prone PCR, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Error prone PCR is described, e.g., in Leung, D.W., et al., Technique, 1 :11-15, 1989) and Caldwell, R. C. & Joyce G.F., PCR Methods Applic, 2:28-33, 1992. Briefly, in such procedures, nucleic acids to be mutagenized are mixed with PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction may be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl2, 0.5 mM MnCl2, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR may be performed for 30 cycles of 94°C for 1 min, 450C for 1 min, and 72°C for 1 min. However, it will be appreciated that these parameters may be varied as appropriate. The mutagenized nucleic acids are cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids is evaluated. Variants may also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized. Clones containing the mutagenized DNA are recovered and the activities of the polypeptides they encode are assessed.
Another method for generating variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, e.g., U.S. Patent No. 5,965,408.
Still another method of generating variants is sexual PCR mutagenesis. In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different but highly related DNA sequence in vitro, as a result of random fragmentation of the DNA molecule based on sequence homology, followed by fixation of the crossover by primer extension in a PCR reaction. Sexual PCR mutagenesis is described, e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751. Briefly, in such procedures a plurality of nucleic acids to be recombined are digested with DNase to generate fragments having an average size of 50-200 nucleotides. Fragments of the desired average size are purified and resuspended in a PCR mixture. PCR is conducted under conditions which facilitate recombination between the nucleic acid fragments. For example, PCR may be performed by resuspending the purified fragments at a concentration of 10-30 ng/:l in a solution of 0.2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5 units of Taq polymerase per 100:1 of reaction mixture is added and PCR is performed using the following regime: 94°C for 60 seconds, 94°C for 30 seconds, 50-550C for 30 seconds, 72°C for 30 seconds (30-45 times) and 720C for 5 minutes. However, it will be appreciated that these parameters may be varied as appropriate. In some aspects, oligonucleotides may be included in the PCR reactions. In other aspects, the Klenow fragment of DNA polymerase I may be used in a first set of PCR reactions and Taq polymerase may be used in a subsequent set of PCR reactions. Recombinant sequences are isolated and the activities of the polypeptides they encode are assessed.
Variants may also be created by in vivo mutagenesis. In some aspects, random mutations in a sequence of interest are generated by propagating the sequence of interest in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways. Such "mutator" strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA. Mutator strains suitable for use for in vivo mutagenesis are described, e.g., in PCT Publication No. WO 91/16427.
Variants may also be generated using cassette mutagenesis. In cassette mutagenesis a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide "cassette" that differs from the native sequence. The oligonucleotide often contains completely and/or partially randomized native sequence. Recursive ensemble mutagenesis may also be used to generate variants.
Recursive ensemble mutagenesis is an algorithm for protein engineering (protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.
In some aspects, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described, e.g., in Delegrave (1993) Biotechnology Res. 11 : 1548-1552. Random and site-directed mutagenesis are described, e.g., in Arnold (1993) Current Opinion in Biotechnology 4:450-455. In some aspects, the variants are created using shuffling procedures wherein portions of a plurality of nucleic acids which encode distinct polypeptides are fused together to create chimeric nucleic acid sequences which encode chimeric polypeptides as described in, e.g., U.S. Patent Nos. 5,965,408; 5,939,250. The invention also provides variants of polypeptides of the invention comprising sequences in which one or more of the amino acid residues (e.g., of an exemplary polypeptide, such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, etc.) are substituted with a conserved or non-conserved amino acid residue (e.g., a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Thus, polypeptides of the invention include those with conservative substitutions of sequences of the invention, e.g., the exemplary sequences of the invention, such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, etc., including but not limited to the following replacements: replacements of an aliphatic amino acid such as Alanine, Valine, Leucine and Iso leucine with another aliphatic amino acid; replacement of a Serine with a Threonine or vice versa; replacement of an acidic residue such as Aspartic acid and Glutamic acid with another acidic residue; replacement of a residue bearing an amide group, such as Asparagine and Glutamine, with another residue bearing an amide group; exchange of a basic residue such as Lysine and Arginine with another basic residue; and replacement of an aromatic residue such as Phenylalanine, Tyrosine with another aromatic residue. Other variants are those in which one or more of the amino acid residues of the polypeptides of the invention includes a substituent group.
Other variants within the scope of the invention are those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide, for example, polyethylene glycol. Additional variants within the scope of the invention are those in which additional amino acids are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide. In some aspects, the variants, fragments, derivatives and analogs of the polypeptides of the invention retain the same biological function or activity as the exemplary polypeptides, e.g., a proteolytic activity, as described herein. In other aspects, the variant, fragment, derivative, or analog includes a proprotein, such that the variant, fragment, derivative, or analog can be activated by cleavage of the proprotein portion to produce an active polypeptide. Optimizing codons to achieve high levels of protein expression in host cells
The invention provides methods for modifying enzyme-encoding nucleic acids to modify codon usage. In one aspect, the invention provides methods for modifying codons in a nucleic acid encoding an enzyme to increase or decrease its expression in a host cell, e.g., a bacterial, insect, mammalian, yeast or plant cell. The invention also provides nucleic acids encoding an enzyme modified to increase its expression in a host cell, enzyme so modified, and methods of making the modified enzymes. The method comprises identifying a "non-preferred" or a "less preferred" codon in enzyme-encoding nucleic acid and replacing one or more of these non-preferred or less preferred codons with a "preferred codon" encoding the same amino acid as the replaced codon and at least one non-preferred or less preferred codon in the nucleic acid has been replaced by a preferred codon encoding the same amino acid. A preferred codon is a codon over- represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell.
Host cells for expressing the nucleic acids, expression cassettes and vectors of the invention include bacteria, yeast, fungi, plant cells, insect cells and mammalian cells. Thus, the invention provides methods for optimizing codon usage in all of these cells, codon-altered nucleic acids and polypeptides made by the codon-altered nucleic acids. Exemplary host cells include gram negative bacteria, such as Escherichia coli; gram positive bacteria, such as any Bacillus (e.g., B. cereus or B. subtilis) or Streptomyces, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris. Exemplary host cells also include eukaryotic organisms, e.g., various yeast, such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines. Thus, the invention also includes nucleic acids and polypeptides optimized for expression in these organisms and species.
For example, the codons of a nucleic acid encoding an enzyme isolated from a bacterial cell are modified such that the nucleic acid is optimally expressed in a bacterial cell different from the bacteria from which the enzyme was derived, a yeast, a fungi, a plant cell, an insect cell or a mammalian cell. Methods for optimizing codons are well known in the art, see, e.g., U.S. Patent No. 5,795,737; Baca (2000) Int. J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12: 185-188; Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect. Immun. 69:7250-7253, describing optimizing codons in mouse systems; Outchkourov (2002) Protein Expr. Purif. 24: 18-24, describing optimizing codons in yeast; Feng (2000) Biochemistry 39:15399-15409, describing optimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif. 20:252- 264, describing optimizing codon usage that affects secretion in E. coli. Transgenic non-human animals
The invention provides transgenic non-human animals comprising a nucleic acid, a polypeptide (e.g., an enzyme or an antibody of the invention), an expression cassette, a vector, a transfected or a transformed cell of the invention. The transgenic non-human animals can be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice, comprising a nucleic acid or protein of the invention as a heterologous or recombinant sequence. These animals can be used, e.g., as in vivo models to study enzyme activity, or, as models to screen for agents that change the enzyme activity in vivo. The coding sequences for the polypeptides to be expressed in the transgenic non-human animals can be designed to be constitutive, or, under the control of tissue-specific, developmental-specific or inducible transcriptional regulatory factors. Transgenic non-human animals can be designed and generated using any method known in the art; see, e.g., U.S. Patent Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and using transformed cells and eggs and transgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g., Pollock (1999) J. Immunol. Methods 231: 147-157, describing the production of recombinant proteins in the milk of transgenic dairy animals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating the production of transgenic goats. U.S. Patent No. 6,211,428, describes making and using transgenic non-human mammals which express in their brains a nucleic acid construct comprising a DNA sequence. U.S. Patent No. 5,387,742, describes injecting cloned recombinant or synthetic DNA sequences into fertilized mouse eggs, implanting the injected eggs in pseudo-pregnant females, and growing to term transgenic mice whose cells express proteins related to the pathology of Alzheimer's disease. U.S. Patent No. 6,187,992, describes making and using a transgenic mouse whose genome comprises a disruption of the gene encoding amyloid precursor protein (APP).
"Knockout animals" can also be used to practice the methods of the invention. For example, in one aspect, the transgenic or modified animals of the invention comprise a "knockout animal," e.g., a "knockout mouse," engineered not to express an endogenous gene, which is replaced with a gene expressing an enzyme of the invention, or, a fusion protein comprising an enzyme of the invention. As noted above, functional knockouts can also be generated using antisense sequences of the invention, e.g., double-stranded RNAi molecules.
Transfienic Plants and Seeds The invention provides transgenic plants and seeds comprising a nucleic acid, a polypeptide (e.g., an enzyme or an antibody of the invention), an expression cassette or vector or a transfected or transformed cell of the invention. The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). The invention also provides methods of making and using these transgenic plants and seeds. The transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with any method known in the art. See, for example, U.S. Patent No. 6,309,872.
Nucleic acids and expression constructs of the invention can be introduced into a plant cell by any means. For example, nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or, the nucleic acids or expression constructs can be episomes. Introduction into the genome of a desired plant can be such that the host's enzyme production is regulated by endogenous transcriptional or translational control elements. The invention also provides "knockout plants" where insertion of gene sequence by, e.g., homologous recombination, has disrupted the expression of the endogenous gene. Means to generate "knockout" plants are well-known in the art, see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao (1995) Plant J 7:359-365. See discussion on transgenic plants, below.
In one aspect, the first step in production of a transgenic plant involves making an expression construct for expression in a plant cell. These techniques are well known in the art. They can include selecting and cloning a promoter, a coding sequence for facilitating efficient binding of ribosomes to mRNA and selecting the appropriate gene terminator sequences. One exemplary constitutive promoter is CaMV35S, from the cauliflower mosaic virus, which generally results in a high degree of expression in plants. Other promoters are more specific and respond to cues in the plant's internal or external environment. An exemplary light-inducible promoter is the promoter from the cab gene, encoding the major chlorophyll a/b binding protein.
In one aspect, the nucleic acid is modified to achieve greater expression in a plant cell. For example, a sequence of the invention is likely to have a higher percentage of A-
T nucleotide pairs compared to that seen in a plant, some of which prefer G-C nucleotide pairs. Therefore, A-T nucleotides in the coding sequence can be substituted with G-C nucleotides without significantly changing the amino acid sequence to enhance production of the gene product in plant cells.
Selectable marker gene can be added to the gene construct in order to identify plant cells or tissues that have successfully integrated the transgene. This may be necessary because achieving incorporation and expression of genes in plant cells is a rare event, occurring in just a few percent of the targeted tissues or cells. Selectable marker genes encode proteins that provide resistance to agents that are normally toxic to plants, such as antibiotics or herbicides. Only plant cells that have integrated the selectable marker gene will survive when grown on a medium containing the appropriate antibiotic or herbicide. As for other inserted genes, marker genes also require promoter and termination sequences for proper function.
In one aspect, making transgenic plants or seeds comprises incorporating sequences of the invention and, optionally, marker genes into a target expression construct (e.g., a plasmid, a phage), along with positioning of the promoter and the terminator sequences. This can involve transferring the modified gene into the plant through a suitable method. For example, a construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. For example, see, e.g., Christou (1997) Plant MoI. Biol. 35: 197-203; Pawlowski (1996) MoI. Biotechnol. 6: 17-30; Klein (1987) Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use of particle bombardment to introduce transgenes into wheat; and Adam (1997) supra, for use of particle bombardment to introduce YACs into plant cells. For example, Rinehart (1997) supra, used particle bombardment to generate transgenic cotton plants. Apparatus for accelerating particles is described U.S. Pat. No. 5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000 particle acceleration instrument; see also, John, U.S. Patent No. 5,608,148; and Ellis, U.S. Patent No. 5, 681,730, describing particle-mediated transformation of gymnosperms. In one aspect, protoplasts can be immobilized and injected with a nucleic acids, e.g., an expression construct. Although plant regeneration from protoplasts is not easy with cereals, plant regeneration is possible in legumes using somatic embryogenesis from protoplast derived callus. Organized tissues can be transformed with naked DNA using gene gun technique, where DNA is coated on tungsten microprojectiles, shot 1/lOOth the size of cells, which carry the DNA deep into cells and organelles. Transformed tissue is then induced to regenerate, usually by somatic embryogenesis. This technique has been successful in several cereal species including maize and rice.
Nucleic acids, e.g., expression constructs, can also be introduced in to plant cells using recombinant viruses. Plant cells can be transformed using viral vectors, such as, e.g., tobacco mosaic virus derived vectors (Rouwendal (1997) Plant MoI. Biol. 33:989- 999), see Porta (1996) "Use of viral replicons for the expression of genes in plants," MoI. Biotechnol. 5:209-221.
Alternatively, nucleic acids, e.g., an expression construct, can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl. Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed. (Springer- Verlag, Berlin 1995). The DNA in an A. tumefaciens cell is contained in the bacterial chromosome as well as in another structure known as a Ti (tumor-inducing) plasmid. The Ti plasmid contains a stretch of DNA termed T-DNA (-20 kb long) that is transferred to the plant cell in the infection process and a series of vir
(virulence) genes that direct the infection process. A. tumefaciens can only infect a plant through wounds: when a plant root or stem is wounded it gives off certain chemical signals, in response to which, the vir genes of A. tumefaciens become activated and direct a series of events necessary for the transfer of the T-DNA from the Ti plasmid to the plant's chromosome. The T-DNA then enters the plant cell through the wound. One speculation is that the T-DNA waits until the plant DNA is being replicated or transcribed, then inserts itself into the exposed plant DNA. In order to use A. tumefaciens as a transgene vector, the tumor-inducing section of T-DNA have to be removed, while retaining the T-DNA border regions and the vir genes. The transgene is then inserted between the T-DNA border regions, where it is transferred to the plant cell and becomes integrated into the plant's chromosomes.
The invention provides for the transformation of monocotyledonous plants using the nucleic acids of the invention, including important cereals, see Hiei (1997) Plant MoI. Biol. 35:205-218. See also, e.g., Horsch, Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803; Thykjaer (1997) supra; Park (1996) Plant MoI. Biol. 32:1135-1148, discussing T-DNA integration into genomic DNA. See also DΗalluin, U.S. Patent No. 5,712,135, describing a process for the stable integration of a DNA comprising a gene that is functional in a cell of a cereal, or other monocotyledonous plant.
In one aspect, the third step can involve selection and regeneration of whole plants capable of transmitting the incorporated target gene to the next generation. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts , pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants from transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins.
After the expression cassette is stably incorporated in transgenic plants, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Since transgenic expression of the nucleic acids of the invention leads to phenotypic changes, plants comprising the recombinant nucleic acids of the invention can be sexually crossed with a second plant to obtain a final product. Thus, the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a plant of the invention and another plant. The desired effects can be enhanced when both parental plants express the polypeptides of the invention. The desired effects can be passed to future plant generations by standard propagation means.
The nucleic acids and polypeptides of the invention are expressed in or inserted in any plant or seed. Transgenic plants of the invention can be dicotyledonous or monocotyledonous. Examples of monocot transgenic plants of the invention are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn). Examples of dicot transgenic plants of the invention are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana. Thus, the transgenic plants and seeds of the invention include a broad range of plants, including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachio, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solatium, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.
In alternative embodiments, the nucleic acids of the invention are expressed in plants which contain fiber cells, including, e.g., cotton, silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca and flax. In alternative embodiments, the transgenic plants of the invention can be members of the genus Gossypium, including members of any Gossypium species, such as G. arboreum;. G. herbaceum, G. barbadense, and G. hirsutum. The invention also provides for transgenic plants to be used for producing large amounts of the polypeptides (e.g., antibodies, enzymes) of the invention. For example, see Palmgren (1997) Trends Genet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing human milk protein beta-casein in transgenic potato plants using an auxin-inducible, bidirectional mannopine synthase (masl',21) promoter with Agrobacterium tumefaciens-mediated leaf disc transformation methods).
Using known procedures, one of skill can screen for plants of the invention by detecting the increase or decrease of transgene mRNA or protein in transgenic plants. Means for detecting and quantitation of mRNAs or proteins are well known in the art.
In one aspect, transgenic plants of the invention are produced by transformation of natural oleaginous plants. The genetically transformed plants of the invention are then reproduced sexually so as to produce transgenic seeds of the invention. These seeds can be used to obtain transgenic plant progeny.
In one aspect, the enzyme gene is operably linked to an inducible promoter. Polypeptides and peptides
The invention provides isolated, synthetic or recombinant polypeptides having a sequence identity (e.g., at least about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or complete sequence identity) to an exemplary sequence of the invention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO: 116, SEQ ID NO:118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO.162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192 and SEQ ID NO: 194. As discussed above, the identity can be over the full length of the polypeptide, or, the identity can be over a region of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues. Polypeptides of the invention can also be shorter than the full length of exemplary polypeptides. In one aspect, the invention provides a polypeptide comprising only a subsequence of a sequence of the invention, exemplary subsequences can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues. In alternative aspects, the invention provides polypeptides (peptides, fragments) ranging in size between about 5 and the full length of a polypeptide, e.g., an enzyme, exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues, e.g., contiguous residues of an exemplary enzyme of the invention.
Peptides of the invention can be useful as, e.g., labeling probes, antigens, toleragens, motifs, enzyme active sites. Polypeptides of the invention also include antibodies capable of binding to an enzyme of the invention.
In one aspect, the invention provides enzymes having at least one of a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity. In one aspect, the invention provides enzymes having decontamination activity. In one aspect, the invention provides enzymes having non-heme chloroperoxidase (nhCPOs) activity. The invention provides methods for making an using these enzyme, e.g., methods for decontamination, bleaching and degradation of lignin, and the like. The following chart describes selected characteristics, including activity profiles
(enzymatic activities) of exemplary nucleic acids and polypeptides of the invention, including sequence identity comparison of the exemplary sequences to public databases; the listed exemplary enzymatic activities were determined based on this sequence identity comparison to the to public databases. Exemplary sequences described in this chart have been subject to a BLAST search (as described in detail, below) against two sets of databases. The first database set is available through NCBI (National Center for Biotechnology Information). All results from searches against these databases are found in the columns entitled "NR Description", "NR Accession Code", "NR Evalue" or "NR Organism". "NR" refers to the Non-Redundant nucleotide database maintained by NCBI. This database is a composite of GenBank, GenBank updates, and EMBL updates. The entries in the column "NR Description" refer to the definition line in any given NCBI record, which includes a description of the sequence, such as the source organism, gene name/protein name, or some description of the function of the sequence. The entries in the column "NR Accession Code" refer to the unique identifier given to a sequence record. The entries in the column "NR Evalue" refer to the Expect value (Evalue), which represents the probability that an alignment score as good as the one found between the query sequence (the sequences of the invention) and a database sequence would be found in the same number of comparisons between random sequences as was done in the present BLAST search. The entries in the column "NR Organism" refer to the source organism of the sequence identified as the closest BLAST hit.
The second set of databases is collectively known as the GENESEQ™ database, which is available through Thomson Derwent (Philadelphia, PA). All results from searches against this database are found in the columns entitled "Geneseq Protein Description", "Geneseq Protein Accession Code", "Geneseq Protein Evalue", "Geneseq DNA Description", "Geneseq DNA Accession Code" or "Geneseq DNA Evalue". The information found in these columns is comparable to the information found in the NR columns described above, except that it was derived from BLAST searches against the GENESEQ™ database instead of the NCBI databases. In addition, this table includes the column "Predicted EC No.". An EC number is the number assigned to a type of enzyme according to a scheme of standardized enzyme nomenclature developed by the Enzyme Commission of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). The results in the "Predicted EC No." column are determined by a BLAST search against the Kegg (Kyoto Encyclopedia of Genes and Genomes) database. If the top BLAST match has an Evalue equal to or less than e"6, the EC number assigned to the top match is entered into the table. The EC number of the top hit is used as a guide to what the EC number of the sequence of the invention might be. The columns "Query DNA Length" and "Query Protein Length" refer to the number of nucleotides or the number amino acids, respectively, in the sequence of the invention that was searched or queried against either the NCBI or Geneseq databases. The columns
"Geneseq or NR DNA Length" and "Geneseq or NR Protein Length" refer to the number of nucleotides or the number amino acids, respectively, in the sequence of the top match from the BLAST search. The results provided in these columns are from the search that returned the lower Evalue, either from the NCBI databases or the GENESEQ™ database. The columns "GENESEQ™ or NR %ID Protein" and "GENESEQ™ or NR %ID DNA" refer to the percent sequence identity between the sequence of the invention and the sequence of the top BLAST match. The results provided in these columns are from the search that returned the lower Evalue, either from the NCBI databases or the GENESEQ™ database. For example, to aid in reading this chart, for the first entry, the polypeptide having a sequence as set forth in SEQ ID NO:2, encoded e.g., by SEQ ID NO:1, based on, inter alia, sequence identity comparison to public databases, has esterase activity based on similarity to : [Solibacter usitatus Ellin6076] Zi|67865127|gb|EAM60129.1 | esterase [Solibacter usitatus Ellin6076]
[INTENTIONALLY LEFT BLANK]
NR Geneseq Geneseq
SEQ Accession Geneseq Protein Protein Protein ID NO: NR Description Code NR Evalue NR Organism Description Accession Code Evalue esterase [Sohbacter usitatus Elhn6076]
Zi|67865127|gb|EAM60129 1| esterase [Sohbacter usitatus Sohbacter usitatus M. xanthus protein 101, 102 Elhn6076] 67927525 2 00E- 18 Ellm6076 sequence, seq id 9726. ABM91009 8.00E-05
Esterase/lipase [Novosphingobmm aromaticivorans DSM 12444] gi|78775471 jgb|EAP39126.1| Novosphingobmm Esterase/lipase [Novosphingobmm aromaticivorans Anti-biofϊlm 103, 104 aromaticivorans DSM 12444] 79039830 7.00E-38 DSM 12444 polypeptide #7. ADR51223 2.00E-50
alpha/beta hydrolase [Sulfitobacter sp EE-36] gi|83846434|gb|EAP84310.1| alpha/beta hydrolase [Sulfitobacter Sulfitobacter sp. Bacterial polypeptide 105, 106 sp EE-36] 83942341 1.00E-87 EE-36 #10001. ADS22773 6.00E-31
Pseudomonas probable hydrolase [Pseudomonas Pseudomonas aeruginosa polypeptide 107, 108 aeruginosa PAOl] 9946723 3.00E-54 aeruginosa PAOl #3. ABO81098 5.00E-55
lysophospholipase [Bacillus clausii Bacillus clausii Bacterial polypeptide
109, 1 10 KSM-K16] 56909430 5.00E-50 KSM-K16 #10001. ADN27202 4.00E-38
esterase [Oceanobacillus iheyensis Oceanobacillus Monoglyceride lipase 111, 112 HTE831] 22775979 8.00E-95 iheyensis HTE831 gene probe. AAR38794 2.00E-85
Amidohydrolase [Solibacter usitatus Ellin6076] gi|67862983!gb!EAM57998.1| Amidohydrolase [Solibacter usitatus Solibacter usitatus M. xanthus protein 113, 114 Ellin6076] 67929705 5.00E-89 Ellin6076 sequence, seq id 9726. ABM95911 2.00E-29
Bifidobacterium hypothetical protein longum NCC2705 ORF Badol_01001377 [Bifidobacterium Bifidobacterium amino acid sequence 115, 116 adolescentis] 85666532 1.00E-35 adolescentis SEQ ID NO:408. ABP65659 1.00E-31
lipase/esterase [uncultured uncultured DNA encoding 117, 118 bacterium] 45775279 9.00E-56 bacterium hydrolase BD423. ABG31301 3.00E-45
Pseudomonas esterase; putative [Burkholderia Burkholderia aeruginosa polypeptide 119, 120 thailandensis E264] 83719676 1.00E-55 thailandensis E264 #3. ABO76311 2.00E-44
COG0657: Esterase/lipase Rubrivivax Anti-biofilm 121, 122 [Rubrivivax gelatinosus PMl] 47575591 1.00E-108 gelatinosus PMl polypeptide #7. ADR51221 9.00E-53
Beta-lactamase [Parvularcula bermudensis HTCC2503] gi|84690539|gb]EAQ16380.1| Beta- Parvularcula lactamase [Parvularcula bermudensis bermudensis Sorangium cellulosum 123, 124 HTCC2503] 84704105 1.00E-121 HTCC2503 protein Qrf 15. AAY58581 3.00E-62
lipase/esterase [uncultured uncultured Anti-biofilm
125, 126 bacterium] 71483580 5.00E-79 bacterium polypeptide #7. ADR51213 1.00E-71 hypothetical protein MaquDRAFT_2080 [Marinobacter aquaeolei VT8] gi|77868012|gb|EAO99284.1t hypothetical protein
MaquDRAFT_2080 [Marinobacter Marinobacter B. circulans putative
129, 130 aquaeolei VT8] 77953059 4.00E-48 aquaeolei VT8 CapJ protein. AED48765 0.56
Alpha/beta hydrolase [Oceanicola batsensis HTCC2597] gi|84389220|gb|EAQ02017.1| Oceanicola Alpha/beta hydrolase [Oceanicola batsensis M. xanthus protein
131, 132 batsensis HTCC2597] 84502408 5.00E-42 HTCC2597 sequence, seq id 9726. ABM94765 3.00E-20
Alpha/beta hydrolase fold [Prosthecochloris aestuarii DSM 271] gi|68240435|gb|EAN22709.1| Alpha/beta hydrolase fold Herbicidally active [Prosthecochloris aestuarii DSM Prosthecochloris polypeptide SEQ ID
133, 134 271] 68552635 3.00E-53 aestuarii DSM 271 NO 2. ABB92272 8.00E-47
carboxylesterase [Ferroplasma acidarmanus Ferl] gij69269015|reflZP_00609613.1| carboxylesterase [Ferroplasma Ferroplasma Anti-biofilm
135, 136 acidarmanus Ferl] 68140665 4.00E-64 acidarmanus Ferl polypeptide #7. ADR51275 1.00E-162
COG0657: Esterase/lipase [Burkholderia pseudomallei S 13] gij67757144|ref]ZP_00496022.1| COG0657: Esterase/lipase [Burkholderia pseudomallei Pasteur] gi|67683522|reflZP_00477610.1| COG0657: Esterase/lipase [Burkholderia pseudomallei 1710a] Burkholderia DNA encoding
137, 138 gi|67670115|refl 67762114 2.00E-45 pseudomallei S 13 hydrolase BD423. ABG31301 2.00E-42
carboxylesterase [Oceanobacillus Oceanobacillus DNA encoding 139, 140 iheyensis HTE831] 22778115 9 00E-93 iheyensis HTE831 hydrolase BD423 ABG31303 4 00E-89
acyl-CoA thioesterase 1 Pseudomonas Prokaryotic essential 141, 142 [Pseudomonas fluorescens Pf-5] 68345928 7 00E-52 fluorescens Pf-5 gene #34740. ABU40366 5.00E-51
COG0400 Predicted esterase Burkholdeπa Prokaryotic essential 143, 144 [Burkholdeπa fungorum LB400] 48785748 1 00E- 108 fungorum LB400 gene #34740 ABU21945 1 00E- 109
Geobacillus carboxylesterase [Geobacillus kaustophilus Sequence of new 145, 146 kaustophilus HTA426] 56381422 1 00E- 142 HTA426 lipase AAP95375 1 00E- 142
serine esterase [Flavobacteπum sp MED217] gi|85830972|gb|EAQ49429 1| serine esterase [Flavobacteπum sp Flavobactenum sp Bacterial polypeptide 147, 148 MED217] 86142220 7 00E-22 MED217 #10001 ADS29693 1 00E- 13
Arylesterase [Methylobacillus flagellatus KT] gi|68212282|reflZP_00564119 1| Arylesterase [Methylobacillus Methylobacillus Anti-biofilm 149, 150 flagellatus KT] 68189034 2 00E-51 flagellatus KT polypeptide #7 ADR51243 1 00E-118 uncultured Anti-biofilm
151, 152 esterase [uncultured bacterium] 66356146 1 00E-112 bacterium polypeptide #7 ADR51279
Homoserine acetyltransferase Idiomarina Caulobacter vibriodes 153, 154 [Idiomarina loihiensis L2TR] 56180267 1.00E-113 loihiensis L2TR Met A RCO00727. ADO80315 4.00E-51
carboxylesterase [Oceanobacillus Oceanobacillus DNA encoding
155, 156 iheyensis HTE831] 22778115 6.00E-93 iheyensis HTE831 hydrolase BD423. ABG31303 1.00E- 142
probable lipase (triacylglycerol Bacillus Staphylococcus lipase) [Bacillus thuringiensis thuringiensis serovar epidermidis Lys-303 157, 158 serovar konkukian str. 97-27] 49329345 9.00E-71 konkukian str. 97-27 lipase PCR primer #2. ADE52005 9.00E-42 putative acetyl-hydrolase [Nocardioides sp. JS614] gi|71156125|gb|EAO06544.1| putative acetyl-hydrolase Nocardioides sp. Anti-biofilm 159, 160 [Nocardioides sp. JS614] 71368123 4.00E-81 JS614 polypeptide #7. ADR51221 6.00E-62
L. pneumophila
Alpha/beta hydrolase fold Dechloromonas protein SEQ ID NO 161, 162 [Dechloromonas aromatica RCB] 71848065 2.00E-89 aromatica RCB 3367. AEB35768 3.00E-51
Esterase/lipase/thioesterase Ralstonia eutropha Anti-biofilm 167, 168 [Ralstonia eutropha JMP134] 72122621 7.00E-84 JMP 134 polypeptide #7. ADR51221 1.00E-90
lysophospholipase; alpha/beta Archaeoglobus hydrolase superfamily Thermococcus venificus SNP6-24LC 169, 170 [Thermococcus kodakarensis KODl] 57159258 1.00E- 124 kodakarensis KODl esterase. AAE38797 1.00E-149
lipase/esterase [uncultured uncultured Acinetobacter 171, 172 bacterium] 45775279 l.OOE-110 bacterium baumannii protein #19. ADA35491 2.00E-59
Acinetobacter Acinetobacter
175, 176 esterase [Acinetobacter lwoffii]. 21070428 1.00E- 165 lwoffii baumannii protein #19. ADA33656 1.00E- 144
Thiomicrospira Archaeoglobus
Patatin [Thiomicrospira denitrificans ATCC venificus esterase 177, 178 denitrificans ATCC 33889] 78777519 1.00E-20 33889 SNP6-24LC. AAW23079 1.00E- 145
Pseudomonas uncultured fluorescens lipase
179, 180 lipase [uncultured Pseudomonas sp.] 51872565 Pseudomonas sp. protein. AAY55925
Symbiobacterium putative lipase [Symbiobacterium thermophilum IAM Anti-biofilm 181, 182 thermophilum IAM 14863] 51856952 1.00E-IOl 14863 polypeptide #7. ADR51213 1.00E- 180
Pseudomonas
Pseudomonas fluorescens lipase
183, 184 lipase [Pseudomonas fluorescens] 83416413 fluorescens protein. AAY55925
lipase/esterase [uncultured uncultured DNA encoding 185, 186 bacterium] 45775279 8.00E-56 bacterium hydrolase BD423. ABG31301 2.00E-45
Xylella fastidiosa Anti-biofilm
187, 188 esterase [Xylella fastidiosa 9a5c] 9106813 LOOE- 129 9a5c polypeptide #7. ADR51259 1.00E-135
Xaa-Pro proline dipeptidase Pseudoalteromonas Alteromonas sp. strain [Pseudoalteromonas haloplanktis haloplanktis JD6.5 OPAA-2 protein 189, 190 TAC125] 76873906 TAC125 SEQ ID NO:2. AAB12697
uncultured DNA encoding
191, 192 esterase [uncultured bacterium] 66356146 3.00E-49 bacterium hydrolase BD423. ABG31307 1.00E- 171
NON-HEME Primer used to amplify CHLOROPEROXIDASE Pseudomonas putida (CHLORIDE PEROXIDASE) (CPO- Streptomyces haloperoxidase 5'
53, 54 T) (CHLOROPEROXIDASE T). 3914445 1.00E- 141 aureofaciens region DNA. ADU77918 2.00E-49
3-oxoadipate enol-lactonase [Vibrio sp. MED222] gi|858359431gb|EAQ54076.1| 3- Bacterial epoxide oxoadipate enol-lactonase [Vibrio sp. Vibrio sp. hydrolase amino acid
75, 76 MED222] 86146232 4.00E-60 MED222 sequence - SEQ ID 4. ADU00270 1.00E- 16
3-oxoadipate enol-lactonase [Vibrio sp. MED222] gi|85835943|gb|EAQ54076.1| 3- oxoadipate enol-lactonase [Vibrio sp. Vibrio sp. M. xanthus protein
77, 78 MED222] 86146232 1.00E-62 MED222 sequence, seq id 9726. ABM96279 4.00E-18
3-oxoadipate enol-lactonase [Vibrio sp. MED222] gi]85835943|gb|EAQ54076.1] 3- oxoadipate enol-lactonase [Vibrio sp. Vibrio sp. M. xanthus protein
83, 84 MED222] 86146232 1.00E-62 MED222 sequence, seq id 9726. ABM96279 4.00E-18
Corynebacterium
COG2021 : Homoserine glutamicum MP protein acetyltransferase [Rubrivivax Rubrivivax sequence SEQ ID 89, 90 gel atinosus PMl] 47572957 3.00E-32 gelatinosus PMl NO:1148. AAB79726 2.00E-28
hypothetical protein [Caulobacter Caulobacter 93, 94 crescentus CB 15] 13421893 1.00E- 105 crescentus CB 15 Peptide epitope #9. ADX40798 0.87
Esterase/lipase [Frankia sp. EAN lpec] gi|68197894|gb|EAN12188.1| Esterase/lipase [Frankia sp. Frankia sp. Anti-biofilm 95, 96 EANlpec] 68234486 9.00E-33 EANlpec polypeptide #7. ADR51261 6.00E-25 acetyl xylan esterase [Thermotoga Thermotoga Anti-biofilm 99, 100 maritima MSB8] 4980565 maritima MS B 8 polypeptide #7. ADR51289
SEQ ID Geneseq DNA Geneseq DNA Predicted EC Query DNA
NO: Geneseq DNA Description Accession Code Evalue Activity Class Number Length Query Protein Lengt
101, 102 HCMV Towne strain UL153 protein. AAT41418 1.8 717 238
Human digestive system antigen 103, 104 genomic sequence SEQ ID NO: 4885. AAK88819 0.14 870 289
Polyketide synthase related ORF19 105, 106 protein, SEQ ID 39. ADQ91713 0.61 Esterase 3... 960 319
Arabidopsis thaliana protein, SEQ ID 107, 108 1971. ADA69763 0.16 Esterase 3.7.1. 1005 334
Human cancer associated sequence 109, 110 HP1-10-003. SEQ ID 12. ADQ97167 0.033 Esterase 3.1.4.39 825 274
Human testicular antigen encoding 11 1, 112 cDNA SEQ ID NO: 746. ABL98314 1.9 Esterase 3.1.1.1 750 249
113, 114 Plant polypeptide, SEQ ID 5546. ADT16798 0.2 3.5.2.7 1233 410
Novel human protein coding sequence 115, 116 #50. ADE71185 0.011 Esterase 1074 357
DNA encoding novel human 117, 118 diagnostic protein #20574. AAS65401 0.17 Esterase 3.1.. 1038 345
DNA encoding novel human 119, 120 diagnostic protein #20574. AAS65401 0.14 Esterase 3.1.. 897 298
M. capsulatus gene #766 for DNA 121, 122 array. ABQ91084 0.58 Esterase 3.1.. 918 305
Rice abiotic stress responsive 123, 124 polypeptide SEQ ID NO:4152. ACL37329 0.2 3.5.2.6 1242 413
R ruber aldehyde dehydrogenase C- 125, 126 terminus #1. ACC59357 9.00E-06 Esterase 3.1.1. 876 291
Klebsiella pneumoniae polypeptide 129, 130 seqid 7178. ABD00035 0.47 Esterase 756 251
Mycobacteriophage DS6A 36.5 Kd 131, 132 major structural protein N-terminus. AAT09311 0.51 Esterase 3.7.1. 813 270
Drosophila melanogaster polypeptide
133, 134 SEQ ID NO 24465. ABL14740 9.9 3.7.1. 990 329
135, 136 Anti-biofilm polypeptide #7. ADR51274 Esterase 3.1.1.1 849 282
Bifidobacterium longum NCC2705 ORF amino acid sequence SEQ ID 137, 138 NO:408. ABQ81847 0.59 Esterase 3.1.. 924 307
139, 140 DNA encoding hydrolase BD423. ABK89958 3.00E-11 Esterase 3.1.1.1 747 248
141, 142 Prokaryotic essential gene #34740. ACA44236 0.43 3.1.2. 699 232
660 219
143, 144 Prokaryotic essential gene #34740. ACA25815 1.00E-156
145, 146 Sequence of new lipase. AAN92401 Esterase 3.1.1.1 744 247
Human mitochondrial DNA sequence 147, 148 SEQ ID NO 9 ADD33495 0.41 Esterase 660 219
149, 150 Anti-biofilm polypeptide #7 ADR51242 Esterase 3.1.2. 642 213
151, 152 Anti-biofilm polypeptide #7 ADR51278 Esterase 3 1.1. 1092 363
153, 154 Bacterial polypeptide #10001. ADS62215 2.00E-04 Esterase 2 3.1.31 1194 397
155, 156 DNA encoding hydrolase BD423. ABK89958 Esterase 3.1.1.1 747 248
Plant full length insert polypeptide 157, 158 seψd 50020. ADX59820 0.88 Esterase 3.1.1.3 1350 449
R ruber aldehyde dehydrogenase C- 159, 160 terminus #1. ACC59357 2.00E-04 Esterase 3.1.. 906 301
Streptomyces avermitilis protein 161, 162 derivative SEQ ID NO: 8. AAH79277 9.9 Esterase 990 329
C glutamicum coding sequence 167, 168 fragment SEQ ID NO: 1935. AAH65102 3.00E-06 Esterase 3.1.. 927 308
Archaeoglobus venifϊcus SNP6-24LC 169, 170 esterase. AAD58911 Esterase 3.1.4.39 789 262
S. spinosa protein fragment encoded 171, 172 by ORF24, SEQ TD 55. AAF88317 0.009 Esterase 3.1.. 891 296
175, 176 Acmetobacter baumanmi protein #19. ADA29530 6.00E-38 Esterase 3.1.. 909 302
Archaeoglobus vemficus esterase 177, 178 SNP6-24LC. AAT79332 Esterase 3.4.21. 780 259
179, 180 Candida cylindracea lipase gene. AAT10419 Esterase 1413 470
181, 182 Anti-biofilm polypeptide #7. ADR51212 Esterase 3.1.1. 939 312
183, 184 Candida cylindracea lipase gene. AAT10419 Esterase 1431 476
DNA encoding novel human
3.1. 900 299 185, 186 diagnostic protein #20574. AAS65401 0.14 Esterase
187, 188 Anti-biofilm polypeptide #7 ADR51258 2 OOE-87 Esterase 3.1.. 1026 341
189, 190 Organophosphorus acid anhydrolase 2. AAX88785 3.00E-22 JGI 3.4.13.9 1287 428
191 , 192 DN A encoding hydrolase BD423 AB K89962 Esterase 3.1.1. 990 329
M xanthus protein sequence, seq id
53, 54 9726 ACL64606 0 13 Glycosidase 1.11.1.10 837 278
Drosophila melanogaster polypeptide 75, 76 SEQ ID NO 24465 ABL05814 2 2 3.1.1.1 876 291
M. xanthus protein sequence, seq id 77, 78 9726. ACL64601 2.2 3.1.1.1 876 291
M. xanthus protein sequence, seq id 83, 84 9726. ACL64601 2.2 3.1.1.1 876 291
Streptomyces clavuligerus orfδpara ?, 90 protein. ADT75281 0.68 2.3.1.31 1056 351
Streptomyces nanchangensis nlmTl 93, 94 protein SeqIDl l. ADX56092 3.1 1209 402
Pseudomonas aeruginosa polypeptide 95, 96 #3. ABD09695 0.58 3.1.. 912 303
99, 100 Anti-biofilm polypeptide #7. ADR51288 Esterase 3.1.1.41 978 325
In alternative aspects: SEQ ID NO: 190 has aminopeptidase and/or metallopeptidase activity; SEQ ID NO: 192 has enzymatic activity against P-F and P-S bonds/G,V agents, and/or can hydrolytically degrade VX; SEQ ID NO: 194 has OPAA activity. "Amino acid" or "amino acid sequence" can include an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules. The terms "polypeptide" and "protein" can include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids. The term "polypeptide" also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides. The peptides and polypeptides of the invention also include all "mimetic" and "peptidomimetic" forms, as described in further detail, below.
In one aspect, the invention provides polypeptides, nucleic acids, infective vehicles (e.g., viruses, phages comprising organophosphoesterase-encoding nucleic acids of the invention), transduced or infected cells or plants and/or transgenic plants to, and methods of using them, e.g., provide self-protecting, pesticide-resistant cells or plants, where in one aspect the polypeptides of the invention act to hydrolyze P-S or P-F bonds and detoxify acetylcholinesterase- or butyrylcholinesterase- inhibitors. The polypeptides of the invention include enzymes in an active or inactive form.
For example, the polypeptides of the invention include proproteins before "maturation" or processing of prepro sequences, e.g., by a proprotein-processing enzyme, such as a proprotein convertase to generate an "active" mature protein. The polypeptides of the invention include enzymes inactive for other reasons, e.g., before "activation" by a post- translational processing event, e.g., an endo- or exo-peptidase or proteinase action, a phosphorylation event, an amidation, a glycosylation or a sulfation, a dimerization event, and the like. Methods for identifying "prepro" domain sequences and signal sequences are well known in the art, see, e.g., Van de Ven (1993) Crit. Rev..Oncog. 4(2): 115-136. For example, to identify a prepro sequence, the protein is purified from the extracellular space and the N-terminal protein sequence is determined and compared to the unprocessed form.
The polypeptides of the invention include all active forms, including active subsequences, e.g., catalytic domains or active sites, of an enzyme of the invention. In one aspect, the invention provides catalytic domains or active sites as set forth below. In one aspect, the invention provides a peptide or polypeptide comprising or consisting of an active site domain as predicted through use of a database such as Pfam (which is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, The Pfam protein families database, A. Bateman, E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S.R. Eddy, S. Griffiths-Jones, K.L. Howe, M. Marshall, and E.L.L. Sonnhammer, Nucleic Acids Research, 30(l):276-280, 2002) or equivalent. The invention includes polypeptides with or without a signal sequence and/or a prepro sequence. The invention includes polypeptides with heterologous signal sequences and/or prepro sequences. The prepro sequence (including a sequence of the invention used as a heterologous prepro domain) can be located on the amino terminal or the carboxy terminal end of the protein. The invention also includes isolated or recombinant signal sequences, prepro sequences and catalytic domains (e.g., "active sites") comprising sequences of the invention.
Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A.K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA. For example, peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifϊeld (1997) Methods Enzymol. 289:3-13) and automated synthesis may be achieved, e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
The peptides and polypeptides of the invention can also be glycosylated. The glycosylation can be added post-translationally either chemically or by cellular biosynthetic mechanisms, wherein the later incorporates the use of known glycosylation motifs, which can be native to the sequence or can be added as a peptide or added in the nucleic acid coding sequence. The glycosylation can be O-linked or N-linked.
The peptides and polypeptides of the invention, as defined above, include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. As with polypeptides of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Thus, in one aspect, a mimetic composition is within the scope of the invention if it has an enzyme activity.
Polypeptide mimetic compositions of the invention can contain any combination of non-natural structural components. In alternative aspect, mimetic compositions of the invention include one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide of the invention can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., -C(=O)-CH2- for -C(=O)-NH-), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY).
A polypeptide of the invention can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below. Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; D- or L- phenylglycine; D- or L- 2 thieneylalanine; D- or L-I, -2, 3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D- (trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p- biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine; D- or L-2- indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R' -N-C-N-R') such as, e.g., 1- cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia- 4,4- dimetholpentyl) carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile derivative (e.g., containing the CN-moiety in place of COOH) can be substituted for asparagine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues. Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclo- hexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O- acetyl tyrosyl species and 3-nitro derivatives, respectively. Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2- chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-V-nitrobenzo-oxa-l^-diazole. Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro- benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4- hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,- dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
A residue, e.g., an amino acid, of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L-confϊguration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D- amino acid, but also can be referred to as the R- or S- form.
The invention also provides methods for modifying the polypeptides of the invention by either natural processes, such as post-translational processing (e.g., phosphorylation, acylation, etc), or by chemical modification techniques, and the resulting modified polypeptides. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. See, e.g., Creighton, T.E., Proteins - Structure and Molecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983). Solid-phase chemical peptide synthesis methods can also be used to synthesize the polypeptides, or fragments thereof, of the invention. Such method have been known in the art since the early 1960's (Merrifield, R. B., J. Am. Chem. Soc, 85:2149-2154, 1963) (See also Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, 111., pp. 11-12)) and have recently been employed in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits have generally utilized the teachings of H. M. Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing peptides upon the tips of a multitude of "rods" or "pins" all of which are connected to a single plate. When such a system is utilized, a plate of rods or pins is inverted and inserted into a second plate of corresponding wells or reservoirs, which contain solutions for attaching or anchoring an appropriate amino acid to the pin's or rod's tips. By repeating such a process step, i.e., inverting and inserting the rod's and pin's tips into appropriate solutions, amino acids are built into desired peptides. In addition, a number of available FMOC peptide synthesis systems are available. For example, assembly of a polypeptide or fragment can be carried out on a solid support using an
Applied Biosystems, Inc. Model 431 A™ automated peptide synthesizer. Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.
Enzymes of the invention The invention provides novel enzymes, e.g., proteins comprising at least about
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or complete sequence identity to exemplary polypeptides of the invention, e.g., a protein having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, etc., (all even numbered sequences from SEQ ID NO: 2 through SEQ ID NO: 194), antibodies that bind them, and methods for making and using them. The polypeptides of the invention can have any enzyme activity.
In alternative aspects, the enzymes of the invention can have modified or new activities as compared to the exemplary enzymes or the activities described herein. For example, the invention includes enzymes with and without signal sequences and the signal sequences themselves. The invention includes immobilized enzymes, anti-enzyme antibodies and fragments thereof. The invention provides proteins for inhibiting enzyme activity, e.g., antibodies that bind to the enzyme active site. The invention includes homodimers and heterocomplexes, e.g., fusion proteins, heterodimers, etc., comprising the enzymes of the invention. The invention includes enzymes having activity over a broad range of high and low temperatures and pH's (e.g., acidic and basic aqueous conditions).
In one aspect, the invention provides methods of generating enzymes having altered (higher or lower) Kcat/Km. In one aspect, site-directed mutagenesis is used to create additional enzyme enzymes with alternative substrate specificities. The can be done, for example, by redesigning the substrate binding region or the active site of the enzyme. In one aspect, enzymes of the invention are more stable at high temperatures, such as 8O0C to 85°C to 9O0C to 950C, as compared to enzymes from conventional or moderate organisms.
Various proteins of the invention have an enzyme activity, under various conditions. The invention provides methods of making enzymes with different catalytic efficiency and stabilities towards temperature, oxidizing agents and pH conditions. These methods can use, e.g., the techniques of site-directed mutagenesis and/or random mutagenesis. In one aspect, directed evolution can be used to produce enzymes with alternative specificities and stability.
The proteins of the invention are used in methods of the invention that can identify enzyme modulators, e.g., activators or inhibitors. Briefly, test samples (e.g., compounds, such as members of peptide or combinatorial libraries, broths, extracts, and the like) are added to enzyme assays to determine their ability to modulate, e.g., inhibit or activate, substrate cleavage. These inhibitors can be used in industry and research to reduce or prevent undesired isomerization. Modulators found using the methods of the invention can be used to alter (e.g., decrease or increase) the spectrum of activity of an enzyme.
The invention also provides methods of discovering enzymes using the nucleic acids, polypeptides and antibodies of the invention. In one aspect, lambda phage libraries are screened for expression-based discovery of enzymes. In one aspect, the invention uses lambda phage libraries in screening to allow detection of toxic clones; improved access to substrate; reduced need for engineering a host, by-passing the potential for any bias resulting from mass excision of the library; and, faster growth at low clone densities.
Screening of lambda phage libraries can be in liquid phase or in solid phase. In one aspect, the invention provides screening in liquid phase. This gives a greater flexibility in assay conditions; additional substrate flexibility; higher sensitivity for weak clones; and ease of automation over solid phase screening.
The invention provides screening methods using the proteins and nucleic acids of the invention involving robotic automation. This enables the execution of many thousands of biocatalytic reactions and screening assays in a short period of time, e.g., per day, as well as ensuring a high level of accuracy and reproducibility (see discussion of arrays, below). As a result, a library of derivative compounds can be produced in a matter of weeks.
The invention includes enzyme enzymes which are non-naturally occurring enzymes having a different enzyme activity, stability, substrate specificity, pH profile and/or performance characteristic as compared to the non-naturally occurring enzyme.
These enzymes have an amino acid sequence not found in nature. They can be derived by substitution of a plurality of amino acid residues of a precursor enzyme with different amino acids. The precursor enzyme may be a naturally-occurring enzyme or a recombinant enzyme. In one aspect, the enzyme variants encompass the substitution of any of the naturally occurring L-amino acids at the designated amino acid residue positions.
Enzyme signal sequences, prepro and catalytic domains
The invention provides signal sequences (e.g., signal peptides (SPs)), prepro domains and catalytic domains (CDs). The SPs, prepro domains and/or CDs of the invention can be isolated or recombinant peptides or can be part of a fusion protein, e.g., as a heterologous domain in a chimeric protein. The invention provides nucleic acids encoding these catalytic domains (CDs), prepro domains and signal sequences (SPs, e.g., a peptide having a sequence comprising/ consisting of amino terminal residues of a polypeptide of the invention). In one aspect, the invention provides a signal sequence comprising a peptide comprising/ consisting of a sequence as set forth in residues 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44 (or a longer peptide) of a polypeptide of the invention.
For example, in one aspect, exemplary signal sequences consist of (or, comprise):
For SEQ ID NO: 104: MKKLFAGFVLFISFYSHA;
For SEQ ID NO: 106: MQSAASILALALALLAGGVWL; For SEQ ID NO: 108: MRKKLVRLLAWIAGLCLLGLVLLVAAFW APSRSV;
For SEQ ID NO:114: MNRLSLVLLSLLLP AAAAA;
For SEQ ID NO: 118: MKRIATA VFLLHAMTSVA VCA;
For SEQ ID NO: 142: MRLFFLFGLFAVLLLDSINATA;
For SEQ ID NO: 150: MLRTLYLILVLMGLFPLS STVMA; For SEQ ID NO: 154: MNKRSLRKCLSAVGVVAILFSVQQVLA;
For SEQ ID NO: 158: MATMMRGASKLLAGMALAVSALTATGEAFA;
For SEQ ID NO: 188: MKHYVIALTTAFLLYT ALPATA;
For SEQ ID NO:76: MSSALFRLMLVAWLVGSGLVRA;
For SEQ ID NO:78: MNAFWNRLAGATLMLALMSTAWA; For SEQ ID NO: 84: MNAFWNRLAGATLMLALMSTAWA;
For SEQ ID NO:90: MVIYRQLLAGILSLSLSLAALA;
For SEQ ID NO:94: MMKPLIA VLALLW ACQDPGFTQA;
For SEQ ID NO:96: MNTSLCAAALAAWVLMPTAMA.
The enzyme signal sequences (SPs), CDs, and/or prepro sequences of the invention can be isolated peptides, or, sequences joined to another enzyme or a nonenzyme polypeptide, e.g., as a fusion (chimeric) protein. In one aspect, the invention provides polypeptides comprising enzyme signal sequences of the invention. In one aspect, polypeptides comprising enzyme signal sequences SPs, CDs, and/or prepro of the invention comprise sequences heterologous to enzymes of the invention (e.g., a fusion protein comprising an SP, CD, and/or prepro of the invention and sequences from another enzyme or a non-enzyme protein). In one aspect, the invention provides enzymes of the invention with heterologous SPs, CDs, and/or prepro sequences, e.g., sequences with a yeast signal sequence. A protein of the invention can comprise a heterologous SP and/or prepro in a vector, e.g., a pPIC series vector (Invitrogen, Carlsbad, CA). In one aspect, SPs, CDs, and/or prepro sequences of the invention are identified following identification of novel enzyme polypeptides. The pathways by which proteins are sorted and transported to their proper cellular location are often referred to as protein targeting pathways. One of the most important elements in all of these targeting systems is a short amino acid sequence at the amino terminus of a newly synthesized polypeptide called the signal sequence. This signal sequence directs a protein to its appropriate location in the cell and is removed during transport or when the protein reaches its final destination. Most lysosomal, membrane, or secreted proteins have an amino-terminal signal sequence that marks them for translocation into the lumen of the endoplasmic reticulum. The signal sequences can vary in length from 13 to 45 or more amino acid residues. Various methods of recognition of signal sequences are known to those of skill in the art. For example, in one aspect, novel enzyme signal peptides are identified by a method referred to as SignalP. SignalP uses a combined neural network which recognizes both signal peptides and their cleavage sites. (Nielsen, et al., "Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites." Protein Engineering, vol. 10, no. 1, p. 1-6 (1997).
It should be understood that in some aspects enzymes of the invention may not have SPs and/or prepro sequences, and/or catalytic domains (CDs). In one aspect, the invention provides polypeptides (e.g., enzymes) lacking all or part of an SP, a CD and/or a prepro domain. In one aspect, the invention provides a nucleic acid sequence encoding a signal sequence (SP), a CD, and/or prepro from one enzyme operably linked to a nucleic acid sequence of a different enzyme or, optionally, a signal sequence (SPs) and/or prepro domain from a non-enzyme protein may be desired.
The invention also provides isolated or recombinant polypeptides comprising signal sequences (SPs), prepro domain and/or catalytic domains (CDs) of the invention and heterologous sequences. The heterologous sequences are sequences not naturally associated with an SP, prepro domain and/or CD. The sequence to which the SP, prepro domain and/or CD are not naturally associated can be on the SP 's, prepro domain and/or CD's amino terminal end, carboxy terminal end, and/or on both ends of the SP and/or CD. In one aspect, the invention provides an isolated or recombinant polypeptide comprising (or consisting of) a polypeptide comprising a signal sequence (SP), prepro domain and/or catalytic domain (CD) of the invention with the proviso that it is not associated with any sequence to which it is naturally associated (e.g., enzyme sequence). Similarly in one aspect, the invention provides isolated or recombinant nucleic acids encoding these polypeptides. Thus, in one aspect, the isolated or recombinant nucleic acid of the invention comprises coding sequence for a signal sequence (SP), prepro domain and/or catalytic domain (CD) of the invention and a heterologous sequence (i.e., a sequence not naturally associated with the a signal sequence (SP), prepro domain and/or catalytic domain (CD) of the invention). The heterologous sequence can be on the 3' terminal end, 5' terminal end, and/or on both ends of the SP, prepro domain and/or CD coding sequence.
The invention provides fusion of N-terminal or C-terminal subsequences of enzymes of the invention (e.g., signal sequences, prepro sequences) with other polypeptides, active proteins or protein fragments. The production of an enzyme of the invention may also be accomplished by expressing the enzyme as an inactive fusion protein that is later activated by a proteolytic cleavage event (using either an endogenous or exogenous protease activity, e.g. trypsin) that results in the separation of the fusion protein partner and the mature enzyme, e.g., enzyme of the invention. In one aspect, the fusion protein of the invention is expressed from a hybrid nucleotide construct that encodes a single open reading frame containing the following elements: the nucleotide sequence for the fusion protein, a linker sequence (defined as a nucleotide sequence that encodes a flexible amino acid sequence that joins two less flexible protein domains), protease cleavage recognition site, and the mature enzyme (e.g., any enzyme of the invention) sequence. In alternative aspects, the fusion protein can comprise a pectate lyase sequence, a xylanase sequence, a phosphatidic acid phosphatase sequence, or another sequence, e.g., a sequence that has previously been shown to be over-expressed in a host system of interest. Any host system can be used (see discussion, above), for example, E. coli or Pichia pastoris . The arrangement of the nucleotide sequences in the chimeric nucleotide construction can be determined based on the protein expression levels achieved with each fusion construct. Proceeding from the 5' end of the nucleotide construct to the 3 ' prime end of the construct, in one aspect, the nucleotide sequences is assembled as follows: Signal sequence/fusion protein/linker sequence/protease cleavage recognition site/ mature enzyme (e.g., any enzyme of the invention) or Signal sequence/pro sequence/mature enzyme/linker sequence/fusion protein. The expression of enzyme (e.g., any enzyme of the invention) as an inactive fusion protein may improve the overall expression of the enzyme's sequence, may reduce any potential toxicity associated with the overproduction of active enzyme and/or may increase the shelf life of enzyme prior to use because enzyme would be inactive until the fusion protein e.g. pectate lyase is separated from the enzyme, e.g., hydrolase or oxidoreductases, or enzymes with decontamination activity.
In various aspects, the invention provides specific formulations for the activation of an enzyme of the invention expressed as a fusion protein. In one aspect, the activation of the enzyme activity initially expressed as an inactive fusion protein is accomplished using a proteolytic activity or potentially a proteolytic activity in combination with an amino-terminal or carboxyl-terminal peptidase.
Glycosylation
The peptides and polypeptides of the invention (e.g., enzymes, antibodies) can also be glycosylated, for example, in one aspect, comprising at least one glycosylation site, e.g., an N-linked or O-linked glycosylation. In one aspect, the polypeptide can be glycosylated after being expressed in a P. pastoris or a S. pombe. The glycosylation can be added post-translationally either chemically or by cellular biosynthetic mechanisms, wherein the later incorporates the use of known glycosylation motifs, which can be native to the sequence or can be added as a peptide or added in the nucleic acid coding sequence.
Hybrid enzymes and peptide libraries
In one aspect, the invention provides hybrid enzymes and fusion proteins, including peptide libraries, comprising sequences of the invention. The peptide libraries of the invention can be used to isolate peptide modulators (e.g., activators or inhibitors) of targets. The peptide libraries of the invention can be used to identify formal binding partners of targets, such as ligands, e.g., cytokines, hormones and the like.
In one aspect, the fusion proteins of the invention (e.g., the peptide moiety) are conformationally stabilized (relative to linear peptides) to allow a higher binding affinity for targets. The invention provides fusions of enzymes of the invention and other peptides, including known and random peptides. They can be fused in such a manner that the structure of the enzymes are not significantly perturbed and the peptide is metabolically or structurally conformationally stabilized. This allows the creation of a peptide library that is easily monitored both for its presence within cells and its quantity. Amino acid sequence variants of the invention can be characterized by a predetermined nature of the variation, a feature that sets them apart from a naturally occurring form, e.g, an allelic or interspecies variation of an enzyme sequence. In one aspect, the variants of the invention exhibit the same qualitative biological activity as the naturally occurring analogue. Alternatively, the variants can be selected for having modified characteristics. In one aspect, while the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed enzyme variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, as discussed herein for example, M 13 primer mutagenesis and PCR mutagenesis. Screening of the mutants can be done using assays of proteolytic activities. In alternative aspects, amino acid substitutions can be single residues; insertions can be on the order of from about 1 to 20 amino acids, although considerably larger insertions can be done. Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70 residues or more. To obtain a final derivative with the optimal properties, substitutions, deletions, insertions or any combination thereof may be used. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances.
The invention provides enzymes where the structure of the polypeptide backbone, the secondary or the tertiary structure, e.g., an alpha-helical or beta-sheet structure, has been modified. In one aspect, the charge or hydrophobicity has been modified. In one aspect, the bulk of a side chain has been modified. Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative. For example, substitutions can be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example an alpha-helical or a beta- sheet structure; a charge or a hydrophobic site of the molecule, which can be at an active site; or a side chain. The invention provides substitutions in polypeptide of the invention where (a) a hydrophilic residues, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine. The variants can exhibit the same qualitative biological activity (i.e. enzyme activity) although variants can be selected to modify the characteristics of the enzymes as needed.
In one aspect, enzymes of the invention comprise epitopes or purification tags, signal sequences or other fusion sequences, etc. In one aspect, the enzymes of the invention can be fused to a random peptide to form a fusion polypeptide. By "fused" or "operably linked" herein is meant that the random peptide and the enzyme are linked together, in such a manner as to minimize the disruption to the stability of the enzyme structure, e.g., it retains enzyme activity. The fusion polypeptide (or fusion polynucleotide encoding the fusion polypeptide) can comprise further components as well, including multiple peptides at multiple loops.
In one aspect, the peptides (e.g., enzyme subsequences) and nucleic acids encoding them are randomized, either fully randomized or they are biased in their randomization, e.g. in nucleotide/residue frequency generally or per position. "Randomized" means that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. In one aspect, the nucleic acids which give rise to the peptides can be chemically synthesized, and thus may incorporate any nucleotide at any position. Thus, when the nucleic acids are expressed to form peptides, any amino acid residue may be incorporated at any position. The synthetic process can be designed to generate randomized nucleic acids, to allow the formation of all or most of the possible combinations over the length of the nucleic acid, thus forming a library of randomized nucleic acids. The library can provide a sufficiently structurally diverse population of randomized expression products to affect a probabilistically sufficient range of cellular responses to provide one or more cells exhibiting a desired response. Thus, the invention provides an interaction library large enough so that at least one of its members will have a structure that gives it affinity for some molecule, protein, or other factor.
Screening Methodologies and "On-line" Monitoring Devices
In practicing the methods of the invention, a variety of apparatus and methodologies can be used to in conjunction with the polypeptides and nucleic acids of the invention, e.g., to screen polypeptides for enzyme activity, to screen compounds as potential activators or inhibitors of an enzyme activity (e.g., for potential drug screening), for antibodies that bind to a polypeptide of the invention, for nucleic acids that hybridize to a nucleic acid of the invention, to screen for cells expressing a polypeptide of the invention and the like. See, e.g., U.S. Patent No. 6,337,187. Capillary Arrays
Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, San Diego, CA, can be used to in the methods of the invention. Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array, including capillary arrays. Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention. Capillary arrays provide another system for holding and screening samples. For example, a sample screening apparatus can include a plurality of capillaries formed into an array of adjacent capillaries, wherein each capillary comprises at least one wall defining a lumen for retaining a sample. The apparatus can further include interstitial material disposed between adjacent capillaries in the array, and one or more reference indicia formed within of the interstitial material. A capillary for screening a sample, wherein the capillary is adapted for being bound in an array of capillaries, can include a first wall defining a lumen for retaining the sample, and a second wall formed of a filtering material, for filtering excitation energy provided to the lumen to excite the sample.
A polypeptide or nucleic acid, e.g., a ligand or a substrate, can be introduced into a first component into at least a portion of a capillary of a capillary array. Each capillary of the capillary array can comprise at least one wall defining a lumen for retaining the first component. An air bubble can be introduced into the capillary behind the first component. A second component can be introduced into the capillary, wherein the second component is separated from the first component by the air bubble. A sample of interest can be introduced as a first liquid labeled with a detectable particle into a capillary of a capillary array, wherein each capillary of the capillary array comprises at least one wall defining a lumen for retaining the first liquid and the detectable particle, and wherein the at least one wall is coated with a binding material for binding the detectable particle to the at least one wall. The method can further include removing the first liquid from the capillary tube, wherein the bound detectable particle is maintained within the capillary, and introducing a second liquid into the capillary tube.
The capillary array can include a plurality of individual capillaries comprising at least one outer wall defining a lumen. The outer wall of the capillary can be one or more walls fused together. Similarly, the wall can define a lumen that is cylindrical, square, hexagonal or any other geometric shape so long as the walls form a lumen for retention of a liquid or sample. The capillaries of the capillary array can be held together in close proximity to form a planar structure. The capillaries can be bound together, by being fused (e.g., where the capillaries are made of glass), glued, bonded, or clamped side-by- side. The capillary array can be formed of any number of individual capillaries, for example, a range from 100 to 4,000,000 capillaries A capillary array can form a micro titer plate having about 100,000 or more individual capillaries bound together
Arrays, or "Biochφs "
Nucleic acids or polypeptides (including en2ymes, peptides or antibodies) of the invention can be immobilized to or applied to an array Arrays can be used to screen for or monitor libraries of compositions (e g , small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention For example, in one aspect of the invention, a monitored parameter is transcript expression of an enzyme gene One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or "biochip." By using an "array" of nucleic acids on a microchip, some or all of the transcripts of a cell can be simultaneously quantified Alternatively, arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of the invention
Polypeptide arrays" can also be used to simultaneously quantify a plurality of proteins The present invention can be practiced with any known "array," also referred to as a "microarray" or "nucleic acid array" or "polypeptide array" or "antibody array" or "biochip," or variation thereof Arrays are geneπcally a plurality of "spots" or "target elements," each target element comprising a defined amount of one or more biological molecules, e g , oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e g , niRNA transcπpts
The terms "array" or "microarray" or "biochip" or "chip" as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface, as discussed in further detail, below
In one aspect, the enzymes are used as immobilized forms Any immobilization method can be used, e g , immobilization upon an inert support such as diethylaminoethyl-cellulose, porous glass, chitin or cells Cells that express enzymes of the invention can be immobilized by cross-linking, e g with glutaraldehyde to a substrate surface
In practicing the methods of the invention, any known array and/or method of making and using arrays can be incorporated in whole or in part, or variations thereof, as descπbed, for example, in U S Patent Nos 6,277,628, 6,277,489, 6,261,776, 6,258,606, 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics Supp. 21:25-32. See also published U.S. patent applications Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765. Antibodies and Antibody-based screening methods
The invention provides isolated, synthetic or recombinant antibodies that specifically bind to an enzyme of the invention. These antibodies can be used to isolate, identify or quantify the enzyme of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention or other related enzymes. The invention also provides antibodies, or "antibody-like" structures (discussed below), having an enzymatic activity of a polypeptide of the invention, e.g., an antibody or "antibody-like" structure of the invention having haloperoxidase activity (e.g., a chloroperoxidase activity), a dehalogenase activity, an oxidoreductase activity, and/or a prolidase or imidodipeptidase activity. The term "antibody" includes a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody." Thus, the invention provides antibodies, including antigen binding sites and single chain antibodies that specifically bind to an enzyme of the invention. In practicing the methods of the invention, polypeptides having an enzyme activity can also be used.
The antibodies can be used in immunoprecipitation, staining, immunoaffϊnity columns, and the like. If desired, nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array of the invention. Alternatively, the methods of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased. Furthermore, the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of the invention.
Methods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, CA ("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, NY (1986); Kohler (1975) Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
Polypeptides or peptides can be used to generate antibodies, which bind specifically to the polypeptides of the invention. The resulting antibodies may be used in immunoaffinity chromatography procedures to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample. In such procedures, a protein preparation, such as an extract, or a biological sample is contacted with an antibody capable of specifically binding to one of the polypeptides of the invention. In immunoaffinity procedures, the antibody is attached to a solid support, such as a bead or other column matrix. The protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of the invention. After a wash to remove non-specifically bound proteins, the specifically bound polypeptides are eluted. The ability of proteins in a biological sample to bind to the antibody may be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding may be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibody to the sample may be detected using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassays, and Western Blots.
Polyclonal antibodies generated against the polypeptides of the invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to a non-human animal. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies which may bind to the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Techniques described for the production of single chain antibodies (see, e.g., U.S.
Patent No. 4,946,778) can be adapted to produce single chain antibodies to the polypeptides of the invention. Alternatively, transgenic mice may be used to express humanized antibodies to these polypeptides or fragments thereof.
Antibodies generated against the polypeptides of the invention (including anti- idiotype antibodies) may be used in screening for similar polypeptides from other organisms and samples. In such techniques, polypeptides from the organism are contacted with the antibody and those polypeptides which specifically bind the antibody are detected. Any of the procedures described above may be used to detect antibody binding. Immobilized polypeptides
In one aspect, polypeptides, including peptides, enzymes and antibodies of the invention, are used as immobilized forms. The immobilized peptides, enzymes and antibodies of the invention can be used, e.g., for decontamination. Any immobilization method or form of support can be used, e.g., arrays, beads, capillary supports and the like, as described above. In one aspect, peptide/ enzyme/ antibody immobilization can occur upon an inert support such as diethylaminoethyl-cellulose, porous glass, chitin or cells.
Alternatively, cells that express peptides, enzymes and antibodies of the invention can be immobilized by cross-linking, e.g. with glutaraldehyde to a substrate surface. Immobilized enzymes of the invention can be prepared containing enzyme bound to a dry, porous particulate hydrophobic support, with a surfactant, such as a polyoxyethylene sorbitan fatty acid ester or a polyglycerol fatty acid ester. The support can be an aliphatic olefϊnic polymer, such as a polyethylene or a polypropylene, a homo- or copolymer of styrene or a blend thereof or a pre-treated inorganic support. These supports can be selected from aliphatic olefϊnic polymers, oxidation polymers, blends of these polymers or pre-treated inorganic supports in order to make these supports hydrophobic. This pre- treatment can comprise silanization with an organic silicon compound. The inorganic material can be a silica, an alumina, a glass or a ceramic. Supports can be made from polystyrene, copolymers of styrene, polyethylene, polypropylene or from co-polymers derived from (meth)acrylates. See, e.g., U.S. Patent No. 5,773,266.
Kits
The invention provides kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, transgenic seeds or plants or plant parts and/or polypeptides (e.g., peptides, enzymes and antibodies) of the invention. The kits can also comprise the decontaminating, neutralizing or detoxifying compositions of the invention. The kits also can contain instructional material teaching/ informing protocols for using the methodologies and the uses of the invention, e.g., for industrial, military, homeland security uses of the invention, as described herein.
Measuring Metabolic Parameters The methods of the invention provide whole cell evolution, or whole cell engineering, of a cell to develop a new cell strain having a new phenotype by modifying the genetic composition of the cell, where the genetic composition is modified by addition to the cell of a nucleic acid, e.g., a enzyme-encoding nucleic acid of the invention. To detect the new phenotype, at least one metabolic parameter of a modified cell is monitored in the cell in a "real time" or "on-line" time frame. In one aspect, a plurality of cells, such as a cell culture, is monitored in "real time" or "on-line." In one aspect, a plurality of metabolic parameters is monitored in "real time" or "on-line." Metabolic parameters can be monitored using the fluorescent polypeptides of the invention (e.g., enzymes of the invention comprising a fluorescent moiety).
Metabolic flux analysis (MFA) is based on a known biochemistry framework. A linearly independent metabolic matrix is constructed based on the law of mass conservation and on the pseudo-steady state hypothesis (PSSH) on the intracellular metabolites. In practicing the methods of the invention, metabolic networks are established, including the:
• identity of all pathway substrates, products and intermediary metabolites
• identity of all the chemical reactions interconverting the pathway metabolites, the stoichiometry of the pathway reactions,
• identity of all the enzymes catalyzing the reactions, the enzyme reaction kinetics,
• the regulatory interactions between pathway components, e.g. allosteric interactions, enzyme-enzyme interactions etc,
• intracellular compartmentalization of enzymes or any other supramolecular organization of the enzymes, and,
• the presence of any concentration gradients of metabolites, enzymes or effector molecules or diffusion barriers to their movement.
Once the metabolic network for a given strain is built, mathematic presentation by matrix notion can be introduced to estimate the intracellular metabolic fluxes if the on- line metabolome data is available. Metabolic phenotype relies on the changes of the whole metabolic network within a cell. Metabolic phenotype relies on the change of pathway utilization with respect to environmental conditions, genetic regulation, developmental state and the genotype, etc. In one aspect of the methods of the invention, after the on-line MFA calculation, the dynamic behavior of the cells, their phenotype and other properties are analyzed by investigating the pathway utilization. For example, if the glucose supply is increased and the oxygen decreased during the yeast fermentation, the utilization of respiratory pathways will be reduced and/or stopped, and the utilization of the fermentative pathways will dominate. Control of physiological state of cell cultures will become possible after the pathway analysis. The methods of the invention can help determine how to manipulate the fermentation by determining how to change the substrate supply, temperature, use of inducers, etc. to control the physiological state of cells to move along desirable direction. In practicing the methods of the invention, the MFA results can also be compared with transcriptome and proteome data to design experiments and protocols for metabolic engineering or gene shuffling, etc. In practicing the methods of the invention, any modified or new phenotype can be conferred and detected, including new or improved characteristics in the cell. Any aspect of metabolism or growth can be monitored.
Monitoring expression of an mRNA transcript In one aspect of the invention, the engineered phenotype comprises increasing or decreasing the expression of an mRNA transcript or generating new transcripts in a cell. This increased or decreased expression can be traced by use of a enzyme-encoding nucleic acid of the invention. mRNA transcripts, or messages, also can be detected and quantified by any method known in the art, including, e.g., Northern blots, quantitative amplification reactions, hybridization to arrays, and the like. Quantitative amplification reactions include, e.g., quantitative PCR, including, e.g., quantitative reverse transcription polymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or "real-time kinetic RT-PCR" (see, e.g., Kreuzer (2001) Br. J. Haematol. 114:313-318; Xia (2001) Transplantation 72:907-914). In one aspect of the invention, the engineered phenotype is generated by knocking out expression of a homologous gene. The gene's coding sequence or one or more transcriptional control elements can be knocked out, e.g., promoters or enhancers. Thus, the expression of a transcript can be completely ablated or only decreased.
In one aspect of the invention, the engineered phenotype comprises increasing the expression of a homologous gene. This can be effected by knocking out of a negative control element, including a transcriptional regulatory element acting in cis- or trans-, or, mutagenizing a positive control element. One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array.
Monitoring expression of a polypeptides, peptides and amino acids
In one aspect of the invention, the engineered phenotype comprises increasing or decreasing the expression of a polypeptide or generating new polypeptides in a cell. This increased or decreased expression can be traced by use of a enzyme or an antibody of the invention. Polypeptides, peptides and amino acids also can be detected and quantified by any method known in the art, including, e.g., nuclear magnetic resonance (NMR), spectrophotometry, radiography (protein radiolabeling), electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffiαsion chromatography, various immunological methods, e.g. immunoprecipitation, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno- fluorescent assays, gel electrophoresis (e.g., SDS-PAGE), staining with antibodies, fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry, Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, and LC-Electrospray and cap-LC- tandem-electrospray mass spectrometries, and the like. Novel bioactivities can also be screened using methods, or variations thereof, described in U.S. Patent No. 6,057,103. Furthermore, as discussed below in detail, one or more, or, all the polypeptides of a cell can be measured using a protein array.
Industrial and Medical Applications
The invention provides many industrial uses and medical applications for the polypeptides (including enzymes, peptides, antibodies) of the invention, e.g., polypeptides having a hydrolase activity, an esterase activity, e.g., an organophosphohydrolase activity (such as an organophosphoesterase activity) or a carboxylesterase activity, a haloperoxidase activity, e.g., a heme-based (hCPO) or a non- heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, including other enzymes with decontamination activity, e.g., for toxin or poison decontamination, including for example nerve agent detoxification or biological agents detoxification or neutralization (e.g., against anthrax an its spores), as described herein. Thus, in alternative aspects, the invention provides compositions and methods for decontamination, e.g., of toxins such as nerve agents, e.g., V agents (VX agent), G agents (sarin, soman, cyclosarin) or H agents (e.g., mustard gases) and/or biological agents, for civilian, military and/or homeland security purposes.
Non-heme chloroperoxidases (nhCPOs)
The invention provides polypeptides having chloroperoxidase activity, including non-heme chloroperoxidase (nhCPOs) activity, or heme-based chloroperoxidase activity, for e.g., bleaching and degradation of lignin or other natural products, s
In one aspect, these enzymes have hypohalite forming, oxygen transfer and perhydro lysis mechanisms of action. Conventional or high throughput assays can be used to determine the activity of an enzyme, e.g. , if it is a heme or non-heme CPO of the invention, e.g., if a polypeptide is within the scope of the invention, as described, for example, in Pelletier (1995) Biochim. Biophys. Acta 1250:149-157 (describes halogenation assay by non-heme chloroperoxidase); Hofmann (1998) J. MoI. Biol. 279:889-900 (describes structure of a non-heme chloroperoxidase); Picard (1997) Angew Chem. Int. Ed. Engl 36: 1196-1199 (describes assay to determine activation of hydrogen peroxide (H2O2) by non-heme chloroperoxidase to form peracetic acid); Pelletier (1994) Microbiology 140:509-516 (described cloning of a non-heme chloroperoxidase).
Gene sequences coding for enzymes with homology to non-heme chloroperoxidases were discovered from environmental DNA libraries, including the exemplary polypeptides having a sequence as set forth in SEQ ID NO:76 (encoded, e.g., by SEQ ID NO:75), SEQ ID NO:78 (encoded, e.g., by SEQ ID NO:77) and SEQ ID NO:86 (encoded, e.g., by SEQ ID NO:85). These enzymes have no metal cofactors but use a serine hydrolase motif (Gly-x-Ser-x-Gly) to perform a halogenation reaction and to form hypohalites from hydrogen peroxide and chloride, bromide or iodide. The electrophiles formed by this mechanism can halogenate organic substrates. The enzymes can also catalyze the transfer of oxygen from hydrogen peroxide to organic substrates. Chemical bleaching of lignin in pulp and paper manufacture uses hypohalites at large excess and creates significant manufacturing and waste removal costs. These novel enzymes can act to catalytically and locally form hypohalite in the paper making process and can substitute for expensive and environmentally burdensome chemical generation of hypohalite.
These novel enzymes also were demonstrated to degrade O,O-diisopropyl S-(2- diisopropylaminoethyl) phosphorothiolate (Tetriso), used as a surrogate substrate in assays of the invention. The nhCPOs of the invention were discovered in an effort aimed at detoxification of butyrylcholinesterase; these enzymes are inhibitors by hydrolysis of their P-S bonds. 179 candidate enzymes derived from environmental library hits were selected from a larger collection (20,000 clones) on the basis of degradation activity toward 10 μM tetriso. The assay was based on tetriso inhibitory activity toward butyrylcholinesterase (BChE) and uses resorufin butyrate as a detection substrate. Enzymatic hydrolysis of resorufϊn butyrate by BChE forms resorufin anion that is detected by its fluorescence emission. Enzyme hits were screened on the basis of fluorescence emission intensity detected in the host cultures containing tetriso degrading activity. Three discrete enzymes (the exemplary polypeptides having a sequence as set forth in SEQ ID NO:76 (encoded, e.g., by SEQ ID NO:75), SEQ ID NO:78 (encoded, e.g., by SEQ ID NO:77) and SEQ ID NO:86 (encoded, e.g., by SEQ ID NO:85)) were ultimately identified with P-F bond hydrolysis activity. The enzyme sequences were found to be homologous to a family of nhCPO enzymes. Self-protecting, pesticide-resistant crop plants
In one aspect, the invention provides polypeptides (including enzymes, peptides, antibodies) of the invention having activity that can be separated into two classes of organophosphoesterases - which, in one aspect, can rapidly detoxify acetylcholinesterase- or butyrylcholinesterase- inhibitors by hydrolyzing P-S or P-F bonds (e.g., inhibitors applied as pesticides). Also provided are the nucleic acids that encode them, including vectors and transgenic cells and plants comprising these organophosphoesterase-encoding nucleic acids. In one aspect the invention provides transgenic cells and plants (and seeds) capable of expressing these enzyme sequences, e.g., in a crop plant, to provide a self- protecting, pesticide-resistant plant, e.g., a self-protecting, pesticide-resistant crop plant. In one aspect the transgenic cell and plant system of the invention provides the plant or cell protection under conditions of pesticide application to maintain robust growth and development of the cell or plant, e.g., a plant crop. The polypeptides having organophosphoesterase activity of the invention can also be directly applied to a cell or a plant, or alternatively, nucleic acids, vectors or infective entities (e.g., phages, viruses, fungi, etc.) can be used to infect or transduce a cell, plant or crop to effect expression of the organophosphoesterase in the cell, plant or crop, thereby generating a self-protecting, pesticide-resistant cell, plant or crop. In one aspect, this expression (e.g., from a vector, phage, virus, fungi, etc.) is inducible. In one aspect, the expression is constitutive. Thus, the invention provides polypeptides, nucleic acids, infective vehicles (e.g., viruses, phages comprising organophosphoesterase-encoding nucleic acids of the invention), transduced or infected cells or plants and/or transgenic plants to, and methods of using them, e.g., provide a self-protecting, pesticide-resistant cell or a plant. While the invention is not limited by any particular mechanism of action, organophosphoesterases of the invention can act to hydro lyze P-S or P-F bonds in inhibitors of acetyl- cholinesterases or butyrylcholinesterases. Thus, in one aspect, the compositions and methods of the invention can be used to detoxify pesticide inhibitors. In one aspect, in contrast to pesticides, the compositions and methods of the invention are non- toxic to the plants they are applied to as they primarily detoxify pesticides. In one aspect, these compositions and methods of the invention are used to detoxify the following pesticides, and analogs and equivalents thereof: Demeton-S, Demeton-S-methyl, Demeton-S-methylsulphon, Demeton-methyl, Parathion, Phosmet, Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos-ethyl, Amiton, Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton, Endothion, Etion, Ethoate- methyl, Formothion, Malathion, Mercarbam, Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone, Prothidathion, Prothoate, Sophamide, Thiometon, Vamidothion and/or Methamidophos.
Decontamination, Neutralization and Detoxification of Toxic Agents In one aspect, the invention provides polypeptides (including enzymes, peptides, antibodies of the invention), and novel mixture/ formulation/ combinations of enzymes (including polypeptides of the invention, known enzymes, or a mixture thereof) for decontamination, neutralization or detoxification (which includes neutralization), e.g., of toxins such as nerve agents, e.g., V agents (VX agent), G agents (sarin, soman, cyclosarin, tabun) or H agents (e.g., mustard gases) and/or biological agents (e.g., anthrax), for civilian, military and/or homeland security purposes. H agents (mustard) and the persistent nerve agent VX contain sulfur molecules that are readily subject to oxidation reactions; thus, the invention provides polypeptides and methods for catalyzing the oxidation of sulfur-containing toxins, such as H agents (e.g., mustard gas). V agents, e.g., VX and the G agents (G nerve agents), contain phosphorus groups that can be hydrolyzed; thus, the invention provides polypeptides and methods for catalyzing the oxidation of phosphorus-containing toxins, such as VX.
Enzymes used to practice the decontamination, neutralization or detoxification compositions (e.g., enzyme mixtures/ formulations/ combinations of the invention) and methods of the invention, including the exemplary combinations described herein for decontamination, neutralization or detoxification of V agents, H agents, G agents and biological agents, also include use of hydrolases, such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, in addition to the described haloperoxidases, e.g., heme-based (hCPO) or non-heme chloroperoxidases, dehalogenases, prolidases, imidodipeptidases and organophosphoric acid anhydrolases.
Toxic agents that can be decontaminated, neutralized and/or detoxified by practicing the compositions and/or methods of the invention include: G- H- V
Figure imgf000157_0001
Figure imgf000157_0003
Figure imgf000157_0002
V agents
In one aspect, the invention provides enzymes, e g , haloperoxidases, including chloroperoxidases (CPOs), and mixtures/ formulations/ combinations of enzymes, for decontamination, neutralization or detoxification of V agents, such as VX, Soviet V-gas (Russian VX), Tetπso or related compounds (see table, below), e g , any phosphorylthiocholine compound Haloperoxidases, e.g , CPOs, degrade all VX stereoisomers by oxidation
Activity of chloroperoxidases of the invention, and of enzymes used to practice the decontamination, neutralization or detoxification formulations and methods of the invention also can include the activity compπsing (the ability to) catalyze the hydrolysis of methylphosphono fluoridates and/or thiophosphoπc esters The compositions (e g., mixtures/ formulations/ combinations of enzymes) and/or methods of the invention can be used agamst.
V-Senes Agents decontaminated by compositions and methods of the invention
Figure imgf000157_0004
VX also is called by the chemical name methylphosphonothioic acid, S-[2-[bis(l- methylethyl)amino]ethyl]- O-ethyl ester, and has the molecular formula CnH26NO2PS and formula weight 267 37 CAS registry number 50782-69-9 VR is a liquid organophosphate nerve agent with an "oily" consistency which is colorless when pure, and is also called O -isobutyl S-(2-diethylammoethyl)methyl phosphothioate, O-isobutyl S-(2-diethylaminoethyl)methyl thiophosphonate, Oisobutyl S-(N,N-diethylammoethyl) methylphosphonothioate, Russian V-gas, Russian VX, RVX Tetπso, also detoxified by the enzymes of the invention, is a VX analogue, with the chemical name O,0-dnsopropyl S-(2-dnsopropylaminoethyl) phosphorothiolate
Toxicity of nerve agents is typically descπbed in 2 ways LCt50 and LD50 LCt50 refers to the inhalational toxicity of the vapor form. "Ct" refers to the concentration of the vapor or aerosol in the air (measured as mg/m ) multiplied by the time the individual is exposed (measured in mmutes) At 10 mg min/m3, VX is the most toxic of the nerve agents (see Table 2) VX also is the least volatile of the nerve agents This characteπstic makes VX a hazard by percutaneous and dermal routes By contrast, G agents tend to volatilize instead of penetrating the skin Summarized
Toxicity and Half- Lives of Nerve Agents
Figure imgf000158_0001
The decontamination, detoxification (neutralization) methods of the invention, including V agent decontamination/ detoxification/ neutralization, can be practiced in conjunction with any known decontamination, detoxification (neutralization) protocol known in the art, e g , as descπbed by Yang (1999) Chemical detoxification of nerve agent VX, Ace Chem Res 32 109-115, Szafraniec (1990) On the Stoichiometry of Phosphonothiolate Ester Hydrolysis, CRDEC-TR-212, July 1990, AD-A250773, Epstein (1974) The kinetics and mechanisms of hydrolysis of phosphonothiolates, Phosphorus, 1974, 4, 157-163, Ketelaar (1956) Metal-catalyzed hydrolysis of thiophosphoπc esters, Nature 177 392-393, Albizo, et al , Hydrolysis of GD and VX, in Proceedmgs, Army Science Conference (16th), Volume 1, 25-27 October 1988, pp. 33-37, AD-A203101; Yang (1993) Perhydrolysis of nerve agent VX, J. Org. Chem. 58:6964-6965; Yang (1992) Decontamination of chemical warfare agents, Chem. Rev. 92:1729-1743; Yang (1990) Oxidative detoxification of phosphonothiolates, J. Am. Chem. Soc. 112(18):6621-6627. For example, in one aspect, the invention provides mixtures/ formulations/ combinations of at least a haloperoxidase and/or a prolidase, imidodipeptidase and/or organophosphoric acid anhydrolase (OPAA) for decontamination, neutralization or detoxification of an V agent.
In one aspect, the prolidase is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 193, or has an amino acid sequence as set forth in SEQ ID NO: 194.
In one aspect, the haloperoxidase is a heme-based peroxidase activity, e.g., an enzyme having heme-based peroxidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively. The enzyme can be a heme- based chloroperoxidase activity encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 1 , or the enzyme can have an amino acid sequence as set forth in SEQ ID NO:2.
In one aspect, the enzymatic activity comprises a non-heme-based peroxidase activity, e.g., the enzyme having non-heme-based chloroperoxidase activity can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID
NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively. In one aspect, any known prolidase, imidodipeptidase and/or organophosphoric acid anhydrolase (OPAA) can be used in combination with any enzyme of this invention (see, e.g., U.S. Patent No:s 6,080,566; 5,928,927; 6,524,834; 6,469,145; 6,838,277), including a prolidase, imidodipeptidase and/or organophosphoric acid anhydrolase (OPAA), or a haloperoxidase of the invention. In one aspect, any known haloperoxidase (e.g., chloroperoxidase) (see, e.g., U.S. Patent No. 6,251,386) can be used in combination with any enzyme of this invention, including a prolidase, imidodipeptidase and/or organophosphoric acid anhydrolase (OPAA) or a haloperoxidase of the invention. In another aspect, hydrolases, such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having this activity, can also be used in the compositions and method of the invention for the decontamination, neutralization or detoxification of V agents, such as VX, Soviet V-gas (Russian VX), Tetriso or related compounds, e.g., any phosphorylthiocho line compound.
H agents
In one aspect, the invention provides enzymes, e.g., haloperoxidases, including chloroperoxidases, and/or dehalogenases, and enzymes having similar activity, and mixtures/ formulations/ combinations of enzymes (including polypeptides of the invention having haloperoxidase and/or dehalogenase activity, and/or known haloperoxidases and dehalogenases), for decontamination or detoxification (which includes neutralization) of H agents, such as mustard gas, which in one form has the formula 1, 1' thiobis [2 chloroethane] bis-(2-chloroethyl) sulphide; or, beta, beta' dichloroethyl sulphide; also known as 2, 2' dichloroethyl sulphide; or, bis (beta- chloroethyl) sulphide; or l-chloro-2 (beta-chlorodiethylthio) ethane; or sulphur mustard, or "yellow cross liquid."
For example, in one aspect, the invention provides mixtures/ formulations/ combinations of at least a haloperoxidase and/or a dehalogenase for decontamination, neutralization or detoxification of an H agent. In one aspect, the dehalogenase has a sequence as set forth in SEQ ID NO:70 (encoded, e.g., by SEQ ID NO:69) and/or SEQ ID NO:92 (encoded, e.g., by SEQ ID NO:91). While the invention is not limited by any particular mechanism of action, in one aspect, the dehalogenases of the invention, and the dehalogenases used to practice the decontamination/ neutralization / detoxification compositions (e.g., formulations) and methods of the invention, act by de-chlorinating an H agent, e.g., a mustard gas.
In one aspect, the haloperoxidase is a heme-based peroxidase activity, e.g., an enzyme having heme-based peroxidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9;
SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively. The enzyme can be a heme- based chloroperoxidase activity encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 1 , or the enzyme can have an amino acid sequence as set forth in SEQ ID NO:2.
In one aspect, the enzymatic activity comprises a non-heme-based peroxidase activity, e.g., the enzyme having non-heme-based chloroperoxidase activity can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO: 65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
In one aspect, any known dehalogenase can be used in combination with any enzyme of this invention, including a dehalogenase or a haloperoxidase of the invention. In one aspect, any known haloperoxidase (e.g., chloroperoxidase) (see, e.g., U.S. Patent No. 6,251,386) can be used in combination with any enzyme of this invention, including a dehalogenase or a haloperoxidase of the invention.
In another aspect, hydrolases, such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having this activity, can also be used in the compositions and method of the invention for the decontamination, neutralization or detoxification of H agents.
G agents
In one aspect, the invention provides enzymes, e.g., prolidases, organophosphoric acid anhydrolases (OPAAs), and enzymes having similar activity, and mixtures/ formulations/ combinations of enzymes (including polypeptides of the invention having prolidase and/or OPAA activity, and/or known prolidases or OPAAs), for decontamination, neutralization or detoxification (which includes neutralization) of G agents - organophosphate nerve agents - such as tabun (GA), sarin (GB), soman (GD), and cyclosarin (GF) (also, see above). Polypeptides having diisopropyl- fluorophosphatase (DFPase) activity, e.g., as encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71, or having an amino acid sequence as set forth in SEQ ID NO:72, can also be used in the compositions (e.g., mixtures/ formulations/ combinations of enzymes) and methods of the invention for decontamination, neutralization or detoxification of G agents.
In one aspect, prolidases and organophosphoric acid anhydrolases (OPAA) of the invention, or known prolidases and/or OPAAs used to practice the invention, catalyze the hydrolysis of organophosphorous compound soman, and in one aspect, also catalyze the hydrolysis of the dipeptide Gly-Pro. Sarin (GB) has the chemical name methylphosphonofluoridic acid, (1- methylethyl) ester, and has the molecular formula C4HiOFO2P and formula weight 140.09. Its Chemical Abstracts Service registry number is 107-44-8.
Sarin and other G agents are rapidly hydrolyzed in basic solutions, e.g., Na2CO3, NaOH, or KOH;1 GB has a half-life of 0.5 minutes at pH 11 at 25EC. The invention can be practiced in conjunction with known means for hydrolyzing G agents, including e.g., known catalysts for GB hydrolysis such as hypochlorite anion (OCl"), several metal ions and their complexes (Cu+2, UO2 +2, ZrO+2, Moθ2 +2, Th+4 ), and iodosobenzoic acid derivatives. Decontamination systems based on this chemistry that can be incorporated into the protocols of this invention include: • solids, powders and solutions containing various types of bleach (NaOCl" or
Ca(OCl")2)
• DS2 (2% NaOH, 70% diethylenetriamine, 28% ethylene glycol monomethyl ether)
• towelettes moistened with NaOH dissolved in water, phenol, ethanol, and ammonia The invention can be practiced in conjunction with known means for hydrolyzing G agents, including e.g., as described by Yang (1992) Decontamination of chemical warfare agents, Chem. Rev. 92: 1729-1743; Gustafson (1962) ... chelate-catalyzed hydrolysis of isopropyl methylphosphonofluoridate (Sarin). J. Am. Chem. Soc. 84:2309- 2316; Hammond (1989) Hydrolysis of toxic organophosphorus compounds by o- iodosobenzoic acid and its derivatives, J. Am Chem. Soc, 111 :7860-7866.
G-series nerve agents share a number of common physical and chemical properties. At room temperature, the G-series nerve agents are volatile liquids, making them a serious risk for exposure by: dermal contact with liquid nerve agent or inhalation of nerve agent vapor. GB is the most volatile of these agents and evaporates at the same rate as water; GD is the next most volatile. Dispersal devices or an explosive blast also can aerosolize nerve agents. Nerve agent vapors are denser than air, making them particularly hazardous for persons in low areas or underground shelters. GB and GD are colorless, while GA ranges from colorless to brown. GB is odorless, while GA and GD smell fruity.
In another aspect, hydrolases, such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having this activity, can also be used in the compositions and method of the invention for the decontamination, neutralization or detoxification of G agents.
Biological agents
In one aspect, the invention provides enzymes, e.g., haloperoxidases, including chloroperoxidases, dehalogenases, prolidases, organophosphoric acid anhydrolases (OPAAs), and enzymes having similar activity, and mixtures/ formulations/ combinations of enzymes (including polypeptides of the invention having similar activity), for decontamination or detoxification (which includes neutralization) of biological agents, e.g., biological warfare agents, including naturally occurring biological agents, e.g., spores and toxins from bacteria, such as Bacillus anthracis. The toxic biological agents neutralized, ameliorated, sequestered or killed by practicing the invention include, e.g., spores from Bacillus anthracis, the bacterium that causes anthrax. Spores of Bacillus anthracis, can survive drought, bitter cold and other harsh conditions for decades, yet still germinate almost instantly to infect and kill once inside an animal or human host. Because anthrax spores may survive traditional drinking water disinfection methods and can attach themselves to the inside surface of water pipes, the compositions of the invention can be used to disinfect water treatment facilities in the unlikely event of the release of anthrax in a water supply. High concentrations of chlorine are effective at killing anthrax: at 5mg/L (a concentration that might be used by treatment systems during periods when drinking water is turbid) 97 percent of spores were killed after one hour; at 10mg/L (similar to a highly chlorinated swimming pool) 99.99 percent were killed, but the chlorine concentration would be too high for the water to be drinkable.
For example, in one aspect, the invention provides mixtures/ formulations/ combinations of at least a haloperoxidase for decontamination, neutralization or detoxification of a biological agent, e.g., spores and toxins from bacteria, such as Bacillus anthracis. In one aspect, the haloperoxidase has a heme-based peroxidase activity, e.g., an enzyme having heme-based peroxidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO:14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively. The enzyme can be a heme- based chloroperoxidase activity encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 1 , or the enzyme can have an amino acid sequence as set forth in SEQ ID NO:2.
In one aspect, the enzymatic activity comprises a non-heme-based peroxidase activity, e.g., the enzyme having non-heme-based chloroperoxidase activity can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively. In one aspect, any known haloperoxidase (e.g., chloroperoxidase) (see, e.g., U.S. Patent No. 6,251,386) can be used in combination with any enzyme of this invention, including a haloperoxidase of the invention.
In another aspect, hydrolases, such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having this activity, can also be used in the compositions and method of the invention for the decontamination, neutralization or detoxification of biological agents, e.g., spores and toxins from bacteria, such as Bacillus anthracis. In one aspect, mixtures/ formulations/ combinations of the invention comprising at least a haloperoxidase also comprise a halite component (e.g., a solution comprising a halite component), e.g. a halite component comprising a chlorite component, such as a sodium chlorite or a sodium iodite (this is an alternative embodiment for all aspects of the invention comprising use of a haloperoxidase, e.g., a CPO, as a decontaminating reagent, whether for biological reagents or for other toxic agents). In one aspect, the enzyme and chlorite component form ClO2 (chlorine dioxide).
Formulations and Equipment
The polypeptides of the invention (e.g., enzymes, peptides, antibodies), and/or the combinations/ mixtures/ formulations of the invention, can be formulated to achieve a desired effect in practicing the decontamination, detoxification (neutralization) methods of the invention. The desired effect can be a therapeutic (e.g., including a detoxifying, neutralizing and/or a decontaminating effect) or a prophylactic (preventive) effect. In one aspect, the polypeptides of the invention are formulated with emulsifiers, stabilizing agents, foaming agents, surfactants, foams, propellants, liposomes, nanostructures and the like, or can be formulated with appropriate compounds to generate emulsions.
In practicing the decontamination, detoxification (neutralization) methods of the invention, and in applying the polypeptides (e.g., enzymes, peptides, antibodies) of the invention, or the combinations/ mixtures/ formulations of the invention, any known application device can be used, for example, any spray or foaming device (e.g., for topical skin application), or use of any inhaler or nebulizer for direct inhalation of an enzyme or a formulation/ combination/ mixture of the invention, e.g., an inhaler or nebulizer as described in U.S. patent publication no. 20060039870.
Any pulmonary delivery device, e.g., an inhaler (e.g., a dry powder inhaler) or nebulizer, which are well known in the art, can be used. Pulmonary delivery devices are well known to provide local effects in the lungs and pulmonary system by delivering active agents, including chemical compounds, antibodies, polypeptides, and proteins.
Pulmonary delivery devices allow for higher bioavailability of an active agent due to the large surface area of the pulmonary epithelium, resulting in lower doses and fewer side effects. Furthermore, pulmonary delivery devices are cost-effective, easy to use, and are non-invasive.
In one aspect, a pulmonary delivery device for practicing the invention is activatable by inhalation; it can automatically dispense active agent (e.g., an enzyme, or a combination formulation, of the invention) upon inhalation. Aerosol containers which contain an enzyme, or a combination formulation, of the invention can be used. In one aspect, these devices can comprise use of propellants.
Devices used to practice the invention (e.g., for pulmonary delivery or topical delivery) can administer a plurality of metered doses in a controlled manner, allowing controlled and consistent dosing of active agents into the subject's skin, mouth, bronchial passages, pulmonary epithelium. Devices used to practice the invention can operate by utilizing a propellant to eject a constant volume of an active agent, which is adsorbed and/or inhaled by the subject.
The formulations of the invention, and/or a device used to practice the invention, can include a surfactant to prevent aggregation of the "active agent" (e.g., comprising an enzyme, or a combination formulation, of the invention). The "active agent" can be dissolved or suspended in solution. Devices used to practice the invention can utilize propellants for simultaneous inhalation (for pulmonary inhalation devices) or surface application (for skin or mouth delivery devices) and activation of active agent. In one aspect, holding chambers, e.g., spacers, are used to store the aerosolized "active agent", eliminating the need for simultaneous activation and inhalation. In one aspect, a device used to practice the invention provides a constant, metered dosage of the active agent to allow for consistent dosing.
Examples of pulmonary delivery devices that can be adapted for use in practicing this invention are described in, e.g., U.S. patent nos. 5,290,539; 6,615,826; 4,349,945; 6,460,537; 6,029,661; 5,672,581; 5,586,550; 5,511,540; U.S. patent publication no.
20060039870.
In another aspect, nebulizers are used to deliver "active agents", e.g., compositions comprising an enzyme, or a combination formulation, of the invention.
Nebulizers can operate by creating a mist, i.e., nebulizing or atomizing, a formulation of the invention in solution, which is inhaled by the subject. The active agent can be dissolved or suspended in solution. The droplets can be created by any method known in the art, including the use of a fan, a vibrating member, or ultrasonic apparatus. Nebulizers can be more "gentle" than inhalers and powered propellant devices used to practice this invention, and are appropriate for individuals unable to use inhalers, such as infants, young children, and individuals that are seriously ill or incapacitated. Examples of nebulizers are described in, e.g., U.S. patent nos. 6,029,661; 6,748,945; 6,530,370; 6,598,602; 6,009,869.
Devices used to practice the invention (e.g., for pulmonary delivery or topical delivery) can administer dry powders which is "eaten" (mouth administered) or inhaled by the subject. To distribute the dry powder, any method known in the art can be used to propel the active agent, including pneumatic systems, powered fans, or mechanical propulsion, e.g., squeezing of the container. An exemplary device can simply upon the inhalation by the subject. In one aspect, active agent are blended with propellants. In another aspect, there is no blending, thus allowing delivery of larger payloads of active agent; see e.g., U.S. patent no. 6,029,661.
In one aspect, devices used to practice the invention generate an aerosolization of a liquid or a dry powder formulation of the invention, e.g., for skin application, mouth administration, or for inhalation into the lung; and in one aspect, a propellant is used. Any propellant can be used, e.g., a chloroflourocarbon, a hydrofluorocarbon, a hydochlorofluorocarbon, or a hydrocarbon, including triflouromethane, dichlorodiflouromethane, dichlorotetrafiioroethanol, and 1,1,1,2-tetraflouroethane, or combinations thereof; see, e.g., propellant formulations as described in U.S. patent no.5,672,581. The present invention also encompasses any methods known in the art for skin, mouth and/or pulmonary administration; e.g., delivery by intradermal or topical administration, intratracheal inhalation, insufflation, or intubation. The present invention can encompass delivery of a formulation of the invention by solution, a powder, or a mist into the mouth, lungs, or onto or into the skin, by a syringe, tube, or similar device. The invention also provides compositions, e.g., pharmaceutical compositions, comprising an enzyme, or a combination formulation, of the invention. A pharmaceutical composition of the invention can comprise an enzyme or a combination formulation of the invention in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Pharmaceutical compositions can also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline, PBS), carbohydrates (e.g., glucose, mannose, sucrose or dextrose), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives. The invention also provides compositions, e.g., pharmaceutical compositions, formulated as a lyophilate, dry powders, solutions, suspensions or slurries for topical application, mouth administration and/or nebulization. In one aspect, particles are suspended or dissolved within a propellant. Dry powders suitable can include amorphous active agents, crystalline active agents and mixtures of both amorphous and crystalline active agents. Dry powder active agents can have a particle size selected to prevent penetration into the alveoli of the lungs, e.g., from about 0.01 μm to about 4 μm, or less than 3 μm, or from about 0.5 μm to about 1 μm, or about 1 μm in diameter. In one aspect, dry powder active agents have a moisture content below about 10% by weight, or below about 5% by weight, or below about 3% by weight. In one aspect, a lyophilized compound of the invention (including only one, or a combination of enzymes)xomprises (is comprised of) 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or more of enzyme.
Dry powders used to deliver enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, can be prepared by spray drying under conditions which result in a substantially amorphous powder. Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, can be dissolved in a physiologically acceptable aqueous buffer, e.g., a citrate buffer having a pH range from about pH 2 to pH 9. Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, can be dissolved at a concentration from 0.01% by weight to 1% by weight, e.g., at about 0.1% to 0.2%.
In one aspect, solutions of the invention are spray dried, and in one aspect, a substantially amorphous powder is generated, e.g., in a conventional spray drier, e.g., as available from a commercial suppliers such as Niro A/S (Denmark), Buchi (Switzerland) and the like. These spray dried powders, e.g., amorphous powders, can be prepared by lyophilization, vacuum drying, or evaporative drying of a solution comprising enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, under conditions to produce an amorphous structure. In one aspect, this formulation of the invention is ground or milled to produce particles within a desired size range. Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, also can be in a crystalline form. Crystalline dry powders can be prepared by grinding or jet milling the bulk crystalline active agent.
Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, also can be with a powder dispersion device to provide more efficient and reproducible delivery of the active agent and to improve handling characteristics of the formulation of the invention. Exemplary excipients used in formulations of the invention include carbohydrates, e.g., monosaccharides such as fructose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, cellobiose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; amino acids, such as glycine, arginine, aspartic acid, glutamic acid, cysteine, lysine, and the like; organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamin hydrochloride, and the like; peptides and proteins such as aspartame, human serum albumin, gelatin, and the like; and alditols, such as mannitol, xylitol, and the like. Compositions of the invention can also comprise lactose, trehalose, raffinose, maitodextrins, glycine, sodium citrate, human serum albumin and mannitol for purposes of which they are well known in the art.
In one aspect, the amount of enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention, to be administered is an amount necessary to deliver a therapeutically effective or prophylactically effective amount to achieve a desired result, whether that be, in alternative aspects, only partial amelioration of symptoms or a complete abatement of symptoms. In practice, amounts and types of formulations will vary widely depending upon the enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention, used; the severity of the condition; the weight of the subject; desired therapeutic effect; nature and amount of toxic agent exposure. In one aspect, the enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention (e.g., comprising a pharmaceutical composition of the invention), is delivered in one or more doses. As noted above, the enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention (e.g., comprising a pharmaceutical composition of the invention), is delivered in aerosol form. In one aspect, a liquid aerosol formulation of the invention comprises compounds of the invention and a dispersing agent in a physiologically acceptable diluent. In one aspect, the dry powder aerosol formulations are a finely divided solid form of enzymes of the invention, or enzyme formulations/ mixtures combinations of the invention and a dispersing agent.
As noted above, the enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention (e.g., comprising a pharmaceutical composition of the invention), can be suspended, dispersed, or dissolved in solution, e.g., comprising a solvent or liquid dispersive medium that contains, for example, water, ethanol, a polyol (e.g. glycerol, propylene glycol or the like), vegetable oils, non-toxic glycerine esters and suitable mixtures thereof. Suitable flowability may be maintained, by generation of liposomes, administration of a suitable particle size in the case of dispersions, or by the addition of surfactants.
As noted above, the enzyme of the invention, or enzyme formulation/ mixture/ combination of the invention (e.g., comprising a pharmaceutical composition of the invention), further comprise antibacterial and/or antifungal agents, e.g., antibiotics, antibodies, nucleic acids, or a chemical, e.g., a paraben, chlorobutanol, or sorbic acid. In one aspect, the compositions of the invention further comprise an isotonic substances, e.g. sugars, buffers and sodium chloride to assure osmotic pressure similar to those of body fluids, e.g., blood.
Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, also can be in the form of a sterile solution. This can be prepared by using an appropriate solvent and /or an excipient (see above), followed by sterile filtering. In the case of sterile powders suitable for use in the preparation of sterile injectable solutions. Alternative preparation methods include drying in vacuum and lyophilization, which provide powdery mixtures of enzymes of the invention, or formulations/ mixtures of the invention, and desired excipients for subsequent preparation of sterile solutions. Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, also can be formulated as appropriate to be compatible with the contaminated surface to be detoxified or decontaminated (or for prophylactic/ preventive application), e.g., a formulation and/or dosage compatible with skin, mucus membranes, eyes, mouth, nasal passages, lungs and the like. In some aspects, enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, are also formulated to be compatible with inanimate objects onto which they can be applied when practicing the compositions and/or methods of the invention, for example, when they are applied to, or are a part of, any application device as described herein, or any product of manufacture, e.g., a gas mask, an air or water filter, a piece of clothing, textile or fabric, a lens, wood, steel, glass, polymers and the like.
Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, also can be formulated as appropriate in time release formulations, e.g., pills, geltabs, nanostructures, hydrogels, capsules, liposomes, e.g., for ingestion, implantation (e.g., subdermal or intradermal), topical application (e.g., as a gel), as a suppository, or injection (e.g., I.V., parenteral, and the like). The time release can be for treatment (including neutralization, detoxification or decontamination purposes) or for prophylactic purposes (e.g., to anticipate, yet neutralize, detoxify or decontaminate exposure). Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, also can be formulated as appropriate for any living or inanimate object; e.g., the compositions and/or methods of the invention can be practiced on humans or animal, including domestic (e.g., pets, zoo or farm animals), experimental or wild animals, including, e.g., mammals, reptiles, fish or fowl, and the like. Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, also can be formulated in appropriate dosages (e.g., in a tablet, a gel, a liposome, a pill, a powder, an aerosol, a capsule, a geltab, a hydrogel, a lotion, an injectable formulation, a nanostructure, and the like), where the duration and frequency of administration is determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. An appropriate dosage and treatment regimen can provide a therapeutic and/or a prophylactic benefit - an amelioration of symptoms or damage to the exposed individual. Various considerations for determining appropriate dosages are described, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's") (e.g., Remington, The Science and Practice of Pharmacy, 21 st Edition, by University of the Sciences in Philadelphia, Editor).
Appropriate dosages can be determined using experimental models and/or clinical trials, e.g., using a minimum dosage that is sufficient to provide effective therapy. Patients can be monitored for therapeutic effectiveness using physical examination, imaging studies, or assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art. Dose adjustments can be made based on the monitoring findings. For example, an individual with exposure to nerve agent is administered a composition of the invention and cessation of symptoms is monitored to determine appropriate timing and amount of dosages. In other aspect, apparatus for sampling for the presence of toxins or agents can be used to determine when to use a composition or method of the invention, or to determine the effectiveness of a composition or method of the invention. For example, a device for detecting the chemical warfare agent VX, as described in U.S. patent application publication no. 20040161856, can be used in the course of practicing the invention.
Enzymes of the invention, or enzyme formulations/ mixtures/ combinations of the invention, also can be formulated for use as anthrax decontamination/ neutralization solutions, e.g., decontaminating a surface material. In one aspect, an anthrax decontamination solution of the invention is applied by foam, spray, mist, fog, or steam.. In one aspect, the compositions of the invention are enhanced by use of ultraviolet light application.
In one aspect, decontamination/ neutralization solutions/ formulations/ mixtures of the invention, e.g., for V agents, G agents, H agents and/or biological agents (e.g., anthrax) or pesticides are used in military defense applications, or, alternatively, for any civilian application, including decontamination (including neutralization of toxicity) of buildings, post offices, ventilation ducts, carpet, clothes and electronic equipment. In one aspect, the decontamination/ neutralization solutions of the invention can be formulated for compatibility with firefighting foams.
The invention also provides equipment comprising the decontamination/ neutralization solutions/ formulations/ mixtures of the invention, for the decontamination and/or neutralization of any piece of equipment, clothing, building, ship, airplane, truck, car, train, container and the like, from any V agents, G agents, H agents, biological agents and/or pesticides, herbicides, insecticides. Because in one aspect the invention provides coating and paints comprising the decontamination/ neutralization solutions/ formulations/ mixtures of the invention that can be applied to any object, the invention also provides any piece of equipment, clothing, building, ship, airplane, truck, car, train, container and the like comprising an enzyme of the invention, or a solution/ formulation/ mixture of the invention.
Exemplary solutions/ formulations/ mixtures of the invention The invention provides decontamination/ neutralization solutions/ formulations/ mixtures comprising mixtures of different classes of enzymes, wherein these enzymes can be the invention, or enzymes of the invention and known enzymes, or novel combinations of known enzymes. In one aspect, the invention provides decontamination/ neutralization solutions/ formulations/ mixtures comprising one, two, three or four or more enzymes of the invention, or enzymes of the invention and known enzymes, or novel combinations of known enzymes. These enzyme combination solutions/ formulations/ mixtures can be used in methods and products of manufacture of the invention for the decontamination, neutralization or detoxification of biological agents, e.g., spores and toxins from bacteria, such as Bacillus anthracis.
Exemplary triple enzyme combinations of the invention
In one aspect, an exemplary decontamination/ neutralization solution/ formulation/ mixture of the invention comprises a mixture of (at least) three enzymes, each in a different class: a dehalogenase, a haloperoxidase, such as a chloroperoxidase, and an organophosphoric acid anhydrolase (OPAA) or a prolidase.
In one aspect, the dehalogenase is a polypeptide having a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
In one aspect, the haloperoxidase is a chloroperoxidase, and in one embodiment, the chloroperoxidase is a heme-based chloroperoxidase, e.g., a polypeptide encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:1, or having an amino acid sequence as set forth in SEQ ID NO:2. In one aspect, the haloperoxidase is a heme-based peroxidase encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively. In one aspect, the haloperoxidase is a non-heme- based chloroperoxidase encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:6l; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID
NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively. In one aspect, the organophosphoric acid anhydrolase (OPAA) is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 193, or having a sequence as set forth in SEQ ID NO: 194. The DFPase can be encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71, or having an amino acid sequence as set forth in SEQ ID NO:72.
In one aspect, this combination/ mixture/ formulation further comprises at least one diisopropylfluorophosphatase (DFPase). Alternatively, the DFPase is exchangeable with (can be used as a substitute for) the OPAA/ prolidase enzyme.
In another aspect, additional enzymes can be added to this exemplary "triple combination, e.g., hydrolases, such as esterases, e.g., cholinesterases, organophosphohydrolases, such as organophosphoesterases, carboxylesterases, diisopropylfluorophosphatases and oxidoreductases, including known enzymes or polypeptides of the invention having any one of these activities.
In one aspect, the "triple combination" dehalogenase, haloperoxidase (e.g., chloroperoxidase) and organophosphoric acid anhydrolase (OPAA) solution/ formulation/ mixture of the invention has the advantage to the user because this composition detoxifies multiple toxic agents in one application: dehalogenase - H agents, such as mustard gas; haloperoxidase - V agents (e.g., VX gas) and biological agents (e.g., bacillus spores); OPAA - G agents (e.g., Sarin). Thus, the "multiple combination" enzyme mixtures of the invention (including the "triple combination") can be effectively used by individuals (e.g., soldiers on the field) without first needing to identify the noxious/ toxic agent.
In one aspect, when the "multiple combination" enzyme mixture of the invention comprises a haloperoxidase enzyme, the composition also comprises a halite component, e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
Exemplary double enzyme combinations of the invention
CPOs and DFPases/ OPAAs
In one aspect, an exemplary decontamination/ neutralization solution/ formulation/ mixture of the invention comprises a mixture of (at least) two enzymes, each in a different class. As with the "triple combination" of enzymes of the invention, discussed above, these "double enzyme combinations of the invention" can detoxify, neutralize or decontaminate multiple toxic agents in one application.
For example, in one aspect, a "double enzyme combination of the invention" comprises at least one diisopropylfluorophosphatase (DFPase) or organophosphoric acid anhydrolase (OPAA) or a prolidase, and at least one haloperoxidase (e.g., chloroperoxidase, or CPO). One embodiment comprises use of all three enzymes (the diisopropylfluorophosphatase (DFPase), organophosphoric acid anhydrolase (OPAA) or prolidase, and haloperoxidase). As noted above, the DFPase and/or OPAA or prolidase are used to decontaminate, neutralize or detoxify G agents. The haloperoxidase (e.g., CPO) are used to decontaminate, neutralize or detoxify V agents, H agents and/or biological agents (e.g., anthrax).
In one aspect, a dehalogenase is also added to this decontamination/ neutralization solution/ formulation/ mixture of the invention for added effect against H agents (e.g., mustard gas) (e.g., to supplement the effect of CPO on H agents).
In one aspect, a cholinesterase is also added to this decontamination/ neutralization solution/ formulation/ mixture of the invention for added effect against on G agents (e.g., to supplement the effect of OPAA or prolidase and/or DFPase on G agents). In one aspect, the polypeptide having diisopropyl-fluorophosphatase (DFPase) activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72. Exemplary haloperoxidases that can be used in this exemplary "double enzyme combination of the invention" are the same as discussed above for the dehalogenase/ haloperoxidase/ OPAA "triple combination" solution/ formulation/ mixture of the invention. In one aspect, the enzyme having organophosphoric acid anhydrolase (OPAA) or prolidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
In one aspect, as with any enzyme mixture of the invention comprising a haloperoxidase enzyme, the composition can also comprise a halite component, e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
DHs and CPOs
In one aspect, an exemplary decontamination/ neutralization solution/ formulation/ mixture of the invention comprises a mixture of (at least) two enzymes: at least one dehalogenase (DH) enzyme and at least one haloperoxidase (e.g., a chloroperoxidase, or CPO). As noted above, dehalogenases and haloperoxidases are used to decontaminate, neutralize, detoxify H agents. Exemplary haloperoxidases that can be used in this exemplary "double enzyme combination of the invention" are the same as discussed above for the dehalogenase/ haloperoxidase/ OPAA "triple combination" solution/ formulation/ mixture of the invention. Exemplary dehalogenases can have a sequence as set forth in SEQ ID NO: 69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
In one aspect, at least one diisopropylfluorophosphatase (DFPase), organophosphoric acid anhydrolase (OPAA) or prolidase and/or cholinesterase (or two, or all three) are also added to the DH and CPO mixture to also decontaminate, neutralize, detoxify G agents. Exemplary DFPase, OPAA or prolidase and/or cholinesterase enzymes that can be used are the same as those used in the other formulations described herein. For example, in one aspect, the polypeptide having diisopropylfluorophosphatase (DFPase) activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO: 72. In one aspect, the enzyme having organophosphoric acid anhydrolase (OPAA) or prolidase activity is encoded by a nucleic acid having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
In one aspect, as with any enzyme mixture of the invention comprising a haloperoxidase enzyme, the composition can also comprise a halite component, e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components.
DHs and OPAAs
In one aspect, an exemplary decontamination/ neutralization solution/ formulation/ mixture of the invention comprises a mixture of (at least) two enzymes: at least one dehalogenase (DH) enzyme and at least one organophosphoric acid anhydrolase (OPAA) or prolidase. As noted above, dehalogenases (and haloperoxidases) are used to decontaminate, neutralize, detoxify H agents (e.g., mustard gas); organophosphoric acid anhydrolases are used to decontaminate, neutralize, detoxify G agents (e.g., Sarin).
In one aspect, this exemplary mixture of the invention can also comprise at least one haloperoxidase to augment the decontamination, neutralization, detoxification of H agents. As with any enzyme mixture of the invention comprising a haloperoxidase enzyme, the composition can also comprise a halite component, e.g., an iodite or a chlorite component; e.g., sodium chlorite or sodium iodite, or equivalent components. In one aspect, this exemplary mixture of the invention can also comprise at least one diisopropylfluorophosphatase (DFPase) and/or cholinesterase enzyme to supplement the OPAA or prolidase decontamination, neutralization, detoxification of G agent.
Exemplary formulations of enzyme combinations of the invention The enzyme combinations of the invention can be formulated individually or collectively in one or more formulations. For example, enzymes used in these decontamination, neutralization, detoxification combinations of the invention can be individually or collectively formulated in any manner, e.g., as described herein, for example, as an edible delivery agent, an injectable liquid, a spray, a tablet, a pill, a gel, a hydrogel, a liposome, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, an aerosol, a powder, a lyophilized compound, a propellant, a foam, an emulsion, a nanostructure or a combination thereof.
In one aspect, a lyophilized compound of the invention (including only one, or a combination of enzymes) comprises (is comprised of) 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or more of enzyme.
Evaluation of symptoms - practicing the invention
In practicing the decontamination, detoxification (neutralization) methods of the invention, and in applying the polypeptides (e.g., enzymes, peptides, antibodies) of the invention, or the combinations/ mixtures/ formulations of the invention, standard clinical/ medical evaluations can be used to determine when to use a composition or method of the invention, or to determine the effectiveness of a composition or method of the invention. For example, for many nerve gas agents, clinical signs and symptoms are related to excessive stimulation at the cholinergic nicotinic and muscarinic receptors. Central effects may be mediated by cholinergic receptors, as well as by effects on N-methyl-D- aspartate-ergic and GABA-ergic systems. A summary of the clinical effects of nerve agents:
Figure imgf000177_0001
Figure imgf000178_0001
Eyes: The most common effects of nerve agents on the eyes are conjunctival injection and pupillary constriction, known as miosis. The patient complains of eye pain, dim vision, and blurred vision. This is most likely from direct contact between the agent and eye. Miosis may persist for long periods and may be unilateral. Severe miosis results in the complaint of dim vision. Ciliary spasm also may cause eye pain. Patients exposed to VX may not have miosis. This is most likely because the eye usually is not exposed directly to the agent, unlike with G agents. Miosis may be a delayed sign of VX exposure. Nose: Rhinorrhea is most common after a vapor exposure but also can be observed with exposures by other routes.
Lungs: Shortness of breath is an important complaint. Patients may describe chest tightness, respiratory distress, or gasping and even may present in apnea. Broncho- constriction and excessive bronchial secretions cause these important life-threatening symptoms. With severe exposures, death may result from central respiratory depression and/or complete paralysis of the muscles of respiration. Respiratory failure is the major cause of death in nerve agent poisoning.
Skeletal muscle: Fasciculations are the most specific identifiable manifestations of intoxication with these agents. Upon initial exposure they can be localized, but they then spread to cause generalized involvement of the entire musculature (after severe exposures). Myoclonic jerks (twitches) may be observed. Eventually, muscles fatigue and a flaccid paralysis ensues.
Skin: With small liquid exposures, localized sweating can be observed with the fasciculations. Generalized diaphoresis can be observed with larger exposures. Gastrointestinal: Abdominal cramping can be observed. With larger exposures, nausea, vomiting, and diarrhea are more prominent.
Heart: The patient may present with either bradycardia or tachycardia. Heart rate depends on the predominance of adrenergic stimulation (resulting in tachycardia) or of the parasympathetic tone (causing bradycardia via vagal stimulation). Heart rate is an unreliable sign of nerve agent poisoning. Many disturbances in cardiac rhythm have been reported after both organophosphate and nerve agent poisonings. Heart blocks and premature ventricular contractions can be observed. The 2 arrhythmias of greatest concern that have been reported include torsade de pointes and ventricular fibrillation.
Central nervous system: Smaller exposures to nerve agents may result in behavioral changes such as anxiety, psychomotor depression, intellectual impairment, and unusual dreams. Large exposures to nerve agents result in loss of consciousness and seizures. Most signs and symptoms are related to the excessive activation and subsequent fatigue at the cholinergic receptor. Some authors have divided exposures into minimal, moderate, and severe toxicity. Signs and symptoms associated with each exposure are
10 summarized:
Severity of Toxicity from Liquid and Vapor Exposures
Figure imgf000179_0001
In one aspect, the invention provides enzymes, e.g., haloperoxidases, including chloroperoxidases, and/or dehalogenases, and mixtures/ formulations/ combinations of 15 enzymes, for decontamination or detoxification (which includes neutralization) of H agents, such as mustard gas. In evaluating acute poisoning by mustard gas: the effects of exposure to mustard gas vapour or liquid are typically delayed for several hours. The delay is shorter in case of liquid contamination. In the first hour after exposure to mustard gas vapour or liquid no signs or symptoms are usually produced, but nausea, 20 retching, vomiting and eye smarting have been occasionally reported.
Exposure to superlethal concentrations may induce convulsions, coma and death within one hour after exposure. Nausea, fatigue, headache, eye inflammation with intense eye pain, lachrymation, blepharospasm, photophobia and rhinorrhoea, followed by reddening of face and neck, soreness of throat and increased pulse and respiratory rate develop at two to six hours post exposure. Six to twenty four hours post exposure the above symptoms are generally increased in severity and are accompanied by skin inflammation followed by blister formation in the warmest areas such as genito-perineal area, buttocks, axillae and on the inner aspects of thighs. In the next twenty four hours the condition generally worsens, blistering becomes more marked, coughing appears. Mucus, pus and necrotic slough may be expectorated. Intense itching of skin and increased skin pigmentation occur. The blood count may reveal anemia and neutropenia four days post exposure. In general, initial leukocytosis on the first 2 to 3 days after exposure is followed by leukopenia in severe intoxicated patients.
A few hours after the ingestion of mustard contaminated food or water, the following signs and symptoms develop: nausea, vomiting, abdominal pain, bloody vomiting and diarrhea with signs of shock and prostration in severe poisoning. The patients who are severely intoxicated may die during the second week after exposure due to respiratory complications and septic shock.
Activities of enzymes of the invention
The following exemplary enzymes of the invention hydrolyze P-F bonds (for reading the chart: e.g., the polypeptide having a sequence as set forth in SEQ ID NO: 102, encoded, e.g., by SEQ ID NO: 101, etc.): SEQ ID NOS:
[INTENTIONALLY LEFT BLANK]
Figure imgf000181_0001
The following exemplary enzymes of the invention hydrolyze P-S bonds (for reading the chart: e.g., the polypeptide having a sequence as set forth in SEQ ID NO:76, encoded, e.g., by SEQ ID NO:79, etc.): SEQ ID NOS:
Figure imgf000181_0002
The following exemplary enzymes of the invention hydrolyze both P-S and P-F bonds (for reading the chart: e.g., the polypeptide having a sequence as set forth in SEQ ID NO: 118, encoded, e.g., by SEQ ID NO:117, etc.):
SEQ ID NOS:
Figure imgf000182_0001
The following exemplary enzymes of the invention can be used for decontamination, including by not limited to crop plant protection and/or for nerve agent detoxification; (for reading the chart: e.g., the polypeptide having a sequence as set forth in SEQ ID NO: 118, encoded, e.g., by SEQ ID NO: 117, which include enzymes active against P-F and P-S bonds and/or G,V agents; etc.):
SEQ ID
NO: Crop plant protection/Nerve Agent Detoxification
117, 1 18 Enzymes active against P-F and P-S bonds/G, V agents
119, 120 Enzymes active against P-F and P-S bonds/G, V agents
127, 128 Enzymes active against P-F and P-S bonds/G,V agents
151, 152 Enzymes active against P-F and P-S bonds/G, V agents
167, 168 Enzymes active against P-F and P-S bonds/G, V agents
171, 172 Enzymes active against P-F and P-S bonds/G,V agents
187, 188 Enzymes active against P-F and P-S bonds/G,V agents
101, 102 Enzymes active against P-F bonds/G agents
103, 104 Enzymes active against P-F bonds/G agents
105, 106 Enzymes active against P-F bonds/G agents
107, 108 Enzymes active against P-F bonds/G agents
109, 110 Enzymes active against P-F bonds/G agents
111, 112 Enzymes active against P-F bonds/G agents
113, 1 14 Enzymes active against P-F bonds/G agents
115, 116 Enzymes active against P-F bonds/G agents
121, 122 Enzymes active against P-F bonds/G agents
123, 124 Enzymes active against P-F bonds/G agents
125, 126 Enzymes active against P-F bonds/G agents
129, 130 Enzymes active against P-F bonds/G agents
131, 132 Enzymes active against P-F bonds/G agents 133, 134 Enzymes active against P-F bonds/G agents
135, 136 Enzymes active against P-F bonds/G agents
137, 138 Enzymes active against P-F bonds/G agents
139, 140 Enzymes active against P-F bonds/G agents
141, 142 Enzymes active against P-F bonds/G agents
143, 144 Enzymes active against P-F bonds/G agents
145, 146 Enzymes active against P-F bonds/G agents
147, 148 Enzymes active against P-F bonds/G agents
149, 150 Enzymes active against P-F bonds/G agents
153, 154 Enzymes active against P-F bonds/G agents
155, 156 Enzymes active against P-F bonds/G agents
157, 158 Enzymes active against P-F bonds/G agents
159, 160 Enzymes active against P-F bonds/G agents
161, 162 Enzymes active against P-F bonds/G agents
163, 164 Enzymes active against P-F bonds/G agents
165, 166 Enzymes active against P-F bonds/G agents
169, 170 Enzymes active against P-F bonds/G agents
173, 174 Enzymes active against P-F bonds/G agents
175, 176 Enzymes active against P-F bonds/G agents
177, 178 Enzymes active against P-F bonds/G agents
179, 180 Enzymes active against P-F bonds/G agents
181, 182 Enzymes active against P-F bonds/G agents
183, 184 Enzymes active against P-F bonds/G agents
185, 186 Enzymes active against P-F bonds/G agents
73, 74 Enzymes active against P-F bonds/G agents
93, 94 Enzymes active against P-F bonds/G agents
95, 96 Enzymes active against P-F bonds/G agents
97, 98 Enzymes active against P-F bonds/G agents
99, 100 Enzymes active against P-F bonds/G agents
75, 76 Enzymes active against P-S bonds/V agents
77, 78 Enzymes active against P-S bonds/V agents
89, 90 Enzymes active against P-S bonds/V agents
The following examples are offered to illustrate, but not to limit the claimed invention.
EXAMPLES Example 1: Exemplary mixtures of oxidoreductases and hydrolases of the invention
The invention provides various mixtures/ combinations/ formulations of oxidoreductases, dehalogenases and hydrolases (including, e.g., haloperoxidases), which can include enzymes of the invention, and also can include mixtures of (new) enzymes of the invention and known enzymes, or can be a novel mixture/ combination using only known enzymes. In various aspects, these mixtures/ combinations/ formulations can provide optimal performance for a desired use, e.g., for decontamination or delignification. In one aspect, a mixture of oxidoreductase and hydrolase enzymes is provided, and this mixture is blended for broad spectrum G-, H-, V- chemical warfare agent and biological warfare agent decontamination.
Summary: The invention provides an enzyme mixture/ formulation/ combinations composition that provides broad-spectrum, agent-specific oxidation and hydrolysis activities as well as hypohalite generation for oxidation or hydrolysis of V-agents, G- agents and H-agents and which generates oxidants such as chlorine dioxide and reactive radicals for killing of biological agents, including anthrax spores. Specifically the exemplary formulation/ mixture of the invention comprises:
(1) a peroxidase, e.g., a haloperoxidase, such as a chloroperoxidase (CPO), which either hydrolyzes (e.g., a non-heme CPO) or oxidizes V- and H-agents (e.g., mustard gases); and,
(2) a dehalogenase which de-chlorinates oxidized mustard H-agents.
In one embodiment, the mixture/ formulation/ combination composition further comprises a third enzyme: (3) a diisopropylfluorophosphatase (DFPase), or equivalent enzyme, which hydrolyzes G-agents.
In one alternative embodiment, a mixture/ formulation/ combination composition comprises a (1) a peroxidase enzyme and a (3) diisopropylfluorophosphatase enzyme.
Enzymes in these exemplary mixtures/ formulations/ combinations can include: (1) Peroxidases, including haloperoxidases (HPOs), such as chloroperoxidases, e.g., heme-based CPOs: these sequences or their homologues:
- Calidariomyces fumago chloroperoxidase (CPO), having a sequence as set forth in SEQ ID NO:2, encoded, e.g., by SEQ ID NO: 1.
- Heme-based peroxidases having a sequence as set forth in SEQ ID NO:3, encoded, e.g., by SEQ ID NO:3.
- Chloroperoxidases (CPOs), including heme-based peroxidases, encoded by a sequence of the invention, e.g., a sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO. l l, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:25, or, comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:26.
- Peroxidases (POs) encoded by a sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO.49, SEQ ID NO:51, or comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO: 50, SEQ ID NO:52.
- Non-heme chloroperoxidases (CPOs) encoded by a sequence of the invention, e.g., a sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65 or SEQ ID NO:67, or comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO.64, SEQ ID NO.66 or SEQ ID NO:68.
(2) dehalogenases whose activity comprises dechlorinating oxidized mustard H- agents, such as dehalogenases encoded by a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO: 69, or comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:70.
(3) diisopropylfluorophosphatases (DFPases) whose activity comprises hydrolyzing G-agents, such as DFPases encoded by a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO.71 (see, e.g., U.S. Patent No. 6,524,834), or comprising an amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:72 (see, e.g., U.S. Patent No. 6,524,834). Assays:
The invention also provides exemplary assays to determine if an enzyme has requisite activity to fall within the scope of the invention, e.g., have requisite activity to be an enzyme in a formulation/ mixture/ combination of the invention:
Assay for agent VX or VX surrogate decontamination by a CPO Time-course of agent VX degradation
1. The environmental library lysates were reconstituted individually with double distilled water (DDW) to their corresponding pre-lyophilized volumes. All steps of the robotic assay were performed in a safety hood. 2. Degradation /reaction 96-well plate: Samples were aliquoted from the lysates suspension at 190 μl in triplicates and 10 μl of 10 μM VX was added (final concentration of VX was lμM). A single well contained only 200 ul lysate without VX (for Eo control of AChE/lysate) and a single well with 200μl DDW (for maxEo AChE control). An "empty" host (a non-transduced host) was used as host control in triplicate. The second control was a triplicate of VX in DDW (maximal AChE inhibition "EI" control). Overall, 22 lysates were examined in each reaction plate.
3. Cold water dilution plate: The VX degradation activity was determined by measuring the residual inhibitory activity of VX at each time interval, by the Ellman method using a SUNRISE™ (Tecan, Research Park, NC) absorbance optical reader. The VX final concentration in the assay plate needed to inhibit human AChE by 95%, within 5 min was 1OnM. At specified time-intervals, lOμl from each well of the reaction plate, were sampled into 90μl cold DDW, for quenching the reaction. Immediately after the cold dilution, 20μl from the water dilution plate were transferred into the Ellman assay plate. 4. Ellman assay plate: The total volume of each well in the assay plate is 200 μl.
20 μl from the dilution plate is sampled into 130μl DTNB solution (final cone, of DTNB - 0.32mM) in the Ellman assay plate, followed by addition of 20μl HuAChE (final AChE activity is 0.03u/ml). The assay plate is incubated for 5 minutes inside the reader at 250C. Following 5 min incubation, 30μl of ATC is added (final conc.-l 12.5μM). The reader, using the Magellan program, is measuring OD/min at λ=405nm, for
1.5min. The final slope is calculated without the first 30 seconds (due to more noisy slope).
5. The stock solutions for the Ellman assay are as follows:
DTNB (Sigma) 0.5mM dissolved in 5OmM phosphate pH=7.5. Human AChE 0.3u/ml dissolved in 5OmM phosphate pH=7.5 with 0.05% BSA and ImM EDTA.
ATC (Acetyl-thiocholine iodide, Sigma) 0.75mM dissolved in DDW. Assay for mustard or mustard surrogate decon by CPO See attached J. Appl Toxicol for protocol
Assay for dehalogenase use in decon Chloride Monitoring during O,O-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate (tetriso) Degradation Using Ion Selective Electrode
Materials:
Accumet Chloride glass body, double junction, ion selective electrode with BNC connector (Fisher Scientific, Pittsburgh, PA)
Accumet Research AR25 Dual Channel pH/Ion Meter (Fisher Scientific) Chloride Standard (note: select a standard that is higher than the expected Cl- production level - 0.100 M Cl- standard works well)
Cl- electrode reference solution (I M Potassium Nitrate, shipped with electrode) - Computer with 9-pin serial port
RS-232 9-pin male to female serial cable
WINWEDGE™ (Adept Scientific) (or other similar serial communication program) Microsoft EXCEL™ (or other similar spreadsheet program) - Stir plate
Protocol:
1. Make a serial dilution of Cl- standard
• This brand of electrode generally has poor response to Cl- concentrations below 100 ppm (2.85 rnM). Keep this in mind while choosing dilutions.
• Use at least three standards.
• The lowest concentration should be at least one order of magnitude lower than the highest concentration
2. Add reference solution to the body of the electrode 3. Connect Cl- electrode to BNC port on back of pH/Ion meter
4. Connect RS-232 cable to meter and computer
5. Standardize meter
• Select "Ion" mode
• On the meter screen, press "Std" to enter standardize mode. • Place lowest concentration of Cl- standard onto stir plate and immerse Cl- electrode into solution. The signal can take up to 10 minutes to fully stabilize - it is important to allow the mV value to stop drifting as much as possible.
• Press "Std" on the meter again to set the standard. After a few moments, enter the standard concentration when prompted. • Repeat these steps for the other standards. Record the displayed mV values for each concentration and enter into a spreadsheet. Plot mV values as a function of log[standard concentration]. The slope of the best fit line should be between -53 and -60. If the slope is outside that range, recalibrate.
6. Immerse the electrode in the tetriso solution. Allow the computer to take a t=0 point without Chloroperoxidase (CPO) in the solution to monitor initial Cl- levels. Start taking readings as CPO is added to the sample.
7. After experiment is over, remove electrode from solution. For short term storage, electrode should be stored in a standard within the general range of interest. Assay for DFPase use in decon
As in Assay for VX or VX surrogate decon by CPO
References Acta Crystallogr. D Biol Crystallogr. 2002 Oct;58(Pt 10 Pt 1): 1757-9.
Biochim Biophys Acta. 2001 Apr 7;1546(2):312-24.
Biochem J. 2001 Feb l;353(Pt 3):579-89.
Protein Expr. Purif. 2001 Feb;21(l):210-9.
Chem Biol Interact. 1993 Jun;87(l-3):17-24 J Appl Toxicol. 2003 Jul-Aug;23(4):225-33.
Example 2: Enzyme Based Active Decontamination
The invention provides novel oxidoreductases, dehalogenases and hydrolases, including mixtures/ combinations/ formulations of enzymes of the invention (which can include enzymes of the invention and/or known enzymes). This example describes the discovery of VX/Tetriso (0,0-diisopropyl S-(2-dϋsopropylaminoethyl) phosphorothiolate) degrading enzymes of the invention from environmental libraries.
(1) Heterologous expression of novel enzymes from environmental libraries:
We have now purified (>20 mgs) of each of the three novel enzymes that were shown to degrade VX and Tetriso using affinity purification. Each of the enzymes has been lyophilized.
- Activity testing: 811 samples (437 different enzymes) are tested for activity on G and V agents. Purified enzymes will be characterized in detail for their activity on VX.
- Assay development. An assay for identifying enzymes that degrade G agents (e.g., having DFPase activity) was developed; a fluoride ion specific electrode was developed by checking its Nernstian response (a Nemstian response occurs when an ion- selective electrode responds according to "local" thermodynamic equilibrium, over a given range of activity or concentration). This probe will be used to compare DFPase, purified organophosphorous hydrolase (OPH) and purified enzymes for surrogate G agent activity. The other assay under optimization is the Ellman assay (a commonly used assay for determining cholinesterase (ChE) activity) for testing detoxification of G and V agents against acetylcholinesterase.
- Characterization of purified proteins: The three purified detoxification hits identified against Tetriso and VX as substrates were incubated at 0.4 mg/ml with DFP (1 mM) and Tetriso (10 μM and 10 mM). The assay worked in the context of positive (DFPase) and negative (boiled DFPase) controls with the DFP, and none of these enzymes showed any activity against this surrogate G agent. However, the controls did not work with Tetriso with our current Ellman assay protocol.
2) Discovery of CPOs from Fungal sources
- Sequence based discovery: The remaining 10 full length clones are being subcloned and made full length.
3. Heterologous expression of Calidariomyces fumaso chloroperoxidase in the host cells E. coli/ Pichia and Saccharomyces:
- A C. fumago chloroperoxidase was subcloned and tested in Saccharomyces and as shown in Figure 5; no protein was detected by western blotting. Figure 5 illustrates an anti-CPO antibody western blot of various Pichia and Saccharomyces transformants showing levels of CPO expression in host cells. Interestingly, in Pichia we do see a band even though we do not see any activity.
4. Optimization of the heterologous expression of C. fumago in C. heterostrophus:
- The optimization of the heterologous expression of C. fumago in C. heterostrophus is at 3 to 4 mg/L of CPO expression in this host.
5. Investigations into CPO inactivation
- Proteomics work support: Since there was concern that samples prepared for analysis may progress beyond the targeted time points (e.g., during thawing of samples), we sought methods of quenching with reductants to neutralize the effects of the oxidant NaClO2. TCEP and sodium sulfite were used in excess immediately following time points taken during the course OfNaClO2 treatment of CPO, then flash frozen and passed on to proteomics for analysis. Complete Tetriso degradation was observed before inactivation of CPO - thus perhaps these mechanisms are unrelated, obviating further analysis of the inactivation of CPO. 6. Investigation into large-scale decontamination of Tetriso
- A series of experiments was designed to determine the amount of chloroperoxidase required to decontaminate 1 wt% Tetriso. Chloroperoxidase-catalyzed oxidation reactions was mechanistically monitored. The following parameters are being monitored: ClO2 " : Iodometric titration with sodium thiosulfate (CIO2 is sparged to avoid interference)
ClO2 : Spectrophotometric analysis of DPD oxidation Tetriso Concentration : GC-MS; pH; Cl" : ISE probe.
Tetriso is gradually degraded in concentrated sodium chlorite (without enzyme), as illustrated in Figure 6A. As illustrated in Figure 6C, the chlorite concentration falls off quickly after 10 to 15 minutes, which then coincides with the ClO2 production, as illustrated in Figure 6D. At the same time chlorine dioxide is produced there is a dramatic drop in pH (as illustrated in Figure 6B) and chloride is consumed. These exemplary methods of the invention can be practiced in the presence of varying amounts of chloroperoxidase. 7. Impact of different buffers on Dehalogenase and DFPase activities
- Dehalogenases were assayed using a bromide probe; and DFPase assayed using a fluoride probe; each in different buffers to determine the impacts of employing different buffers on enzyme performance.
-Dehalogenase: As illustrated in Figure 7, bromide ion formation from dehalogenase-catalyzed hydrolysis of dibromomethane was assayed as a function of enzyme concentration in various buffers. Rates were monitored using a bromide selective electrode. The results of this experiment (data illustrated in Figure 7) indicate that the dehalogenase performs best at pH 7.5 than at lesser pH values, but that there is no dramatic change within the overall pH range. There was also very little difference between the different buffers that were used.
- DFPase: The same experiment (as that illustrated in Figure 7 for dehalogenase) was performed with DFPase, as shown in Figure 8. As illustrated in Figure 8, the rate of fluoride produced by DFPase-catalyzed hydrolysis of DFP (3 mM) was assayed as a function of DFPase concentration in various buffers. Rates were measured using a fluoride selective electrode. The results are very similar to those seen for the dehalogenase. The rates are better at higher pH values but no dramatic changes in DFPase activity are seen within the pH range and there is not a significant change seen between the buffers.
8. Impact of different buffers to resist pH change - The capacity of various buffers to resist acidic pH change was assayed. In light of these studies, at least under these conditions, a pH of greater than 6.5 must be maintained throughout the decontamination reaction. The desirable attributes of a good buffer would be the following: a) Solid components used to self buffer b) pKa within one pH unit of the desired range c) Inexpensive d) High buffer capacity per unit mass
The following experiment was set up to try and address these desirable attributes; the respective buffer solutions were prepared at 300 mM and varying pH. A titrator was used to add 0.1 M HCl over a period of time while monitoring the pH. The results are shown in Figure 9. In Figure 9, the capacity of various buffers to resist pH change after titration with standardized 0.1 M HCl was assayed. Each buffer was tested at three start pH 6.5, 7, and 7.5. Relative strength within pH range =
Phosphate - citrate > Hepes > Phosphate > Tris Mass required to prepare buffer =
Tris < Phosphate - citrate < Phosphate < Hepes (37) (41) (42) (72) Relative cost =
Tris < Phosphate < Phosphate - citrate < Hepes Overall preference =
Phosphate - citrate > Phosphate > Hepes > Tris
9. Corrosion tests Standardized methods for examining the effect of decontamination (decon) solutions on different military-specified paints was tested. The overall protocol is dictated based upon a military protocol, in which pencils of different hardness ratings are used to gauge the resistance of paint coatings to scratching or gouging.
Although this military protocol specifies the method in which to perform the test, it provides no guidelines on the recommended dry film thicknesses (dft) to be used with the test. After several test plates were painted, it was determined that the coatings initially used were too thick (~10 mils, where 1 mil = 0.001"), requiring extended time to dry each coat. At this point, specification sheets were located for each type of paint to be used in this round of tests. Examination of these documents revealed that the recommended dft for the types of paint used range between 1 and 4 mils.
Using this information, another series of test panels was coated with MIL-24441 epoxy polyamide green primer (a non-Volatile Organic Compound (VOC) compliant, two coat, epoxy polyamide system, formulated for immersion service and to protect surfaces from environmental attack) at a thickness of 5 wet mils (approximately 2.5 mils dry). The initial coating was patchy; further coats of primer must be applied to even paint spreading before the enamel top layer can be applied.
Additional studies: 1 (or 2) additional coats of primer will be applied to test plates: 5 to 6 hours (h) after the final coat of primer, an enamel top coat will be applied while the final primer coat is still tacky. The enamel coated plates will be placed in the 600C oven for approximately 2 days to force cure. At this point, Sandia foam (a decontamination formulation that rapidly neutralizes chemical and biological agents) (Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy), 300 mM NaClO2, and 1000 ppm ClO2 will be applied to the plates and the effects on paint hardness will be examined by using pencil testing.
10. Kinetics of VX degradation by chemical hydrolysis
- The screening of environmental library lysates resulted in the discovery of nine VX degradation hits; clones are being enriched, proteins expressed and purified. Since robotic screening of the library was performed without the addition of any oxidizing co- substrate (e.g. NaClO2 or H2O2), VX degradation in these library hits may arise from enzymatic hydrolysis. Preliminary sequence analysis revealed homology with certain known hydrolases.
In order to evaluate the expected maximal rate of enzymatic hydrolysis with the newly discovered hydrolases, the rate of VX chemical hydrolysis was measured at different NaOH concentrations. Figures 10 and 11 (see discussion below) describe the time-course of VX degradation at various NaOH concentrations using either 10 μM or 0.5 mM VX, respectively. The rate of VX degradation was determined by following the residual inhibition of AChE using the Ellman method. At 0.01N NaOH, VX at 10 μM and 5 mM was degraded after 22 h at 11% and 13% respectively. Scheme 1 describes the various possible pathways in VX hydrolysis; the first stage in basic hydrolysis is the attack of hydroxide ion on the phosphorus atom forming a pentacoordinate phosphorus intermediate (I):
Figure imgf000193_0001
Scheme 1 : Pentacoordinate P intermediate (I) Then, there are three possible bond cleavage pathways; the first, in which the P-S bond is cleaved producing the methyl ethyl phosphonic acid and N,N-diisopropylamino ethanethiol leading to complete detoxification of VX:
Figure imgf000194_0001
In the second, ethoxide is expelled to give a toxic compound (so-called "EA2192"):
Figure imgf000194_0002
VX could also hydrolyze via displacement of thiophosphonate anion from a carbon atom also leading to its detoxification:
Figure imgf000194_0003
The nature of the hydrolytic products of VX depends primarily on the pH of the medium. It was noted that at pH>10 the only products are the free thiol and methyl ethyl phosphonic acid (see, e.g., Epstein (1974) The kinetics and mechanisms of hydrolysis of phosphonothiolates in dilute aqueous solution, Phosphorus 4:157-163). Indeed, at all NaOH concentrations measured (except for 0.01 N NaOH where we followed the hydrolysis up to 48 hr only) we obtained a complete detoxification of VX as measured by the AChE inhibition assay, as illustrated in Figures 10 and 11. These kobS values may serve as a frame of reference for the kinetic values that will be obtained with the purified enzymes.
For the data illustrated by Figure 10: Time-Course of VX Degradation by NaOH (VX 10 μM 250C):
Figure imgf000194_0004
For the data illustrated by Figure 11 : Time-Course of VX Degradation by NaOH
(VX 5 mM 250C):
Figure imgf000195_0002
11. Exemplary screening assays using fluorescent substrates
Studies toward finding the optimal fluorescent substrate were initiated, and conditions for screening environmental libraries for VX or GF degradation activity also were initiated. The fluorescent substrates studied: resoruiϊn butyrate (RB), resorufin acetate (RA) and 7- acetoxy-N-methyl quinolinium iodide (AMQI). Figure 12 describes the concentration dependence of BChE inhibition (5 min inhibition of horse serum BChE, Sigma, 2.5 U/ml at 0.2 ml total volume in 96-well plates) by VX and GF using either RB or butyrylthiocholine (BTC) as substrates. BChE activity was measured either by the change in relative fluorescence units with time (RFU/min) using RB as substrate (75 μM) or by following the slope of OD/min at 412 nm using BTC as substrate and DTNB as a thiol reagent (Ellman method).
In Figure 12, data shows the dependence of BChE activity on VX and GF concentrations. The concentration dependence curves of BChE inhibition by either VX or GF, obtained either with RB (fluorescence) or BTC (absorbance) as substrates, were practically identical. Since AChE is inhibited by GF and VX at lower concentrations than BChE (ca., or approximately, 10- to 20-fold lower IC50 values), additional studies will address a broader dynamic range for VX and GF concentrations with AChE inhibition by using the appropriate fluorescent substrate.
10. Decontamination of attenuated B. anthracis spores
The invention also provides compositions and methods for decontamination of biological agents, e.g., biological warfare agents, e.g., B. anthracis spores. Two different mixtures of CPOZNaClO2 in 1 ml total volume were tested: CPO was added repeatedly (5 times) at 2 or 10 units per dose into phosphate buffer (50 mM, pH 7.5) containing NaClO2
(25 mM). CPO-generated oxidizing radicals concentrations were determined by dihydro- pyrimidine dehydrogenase (DPD) (DPD assay, see, e.g., Liem (2002) Clin. Biochem. 35(3):181-187) at 10 min intervals up to 60 min, as illustrated in Figure 13. Figure 13 illustrates studies describing CPO-generated oxidizing radicals concentrations as determined by DPD assay. This data implies that CPO-generated oxidizing radicals levels remained stable for at least 60 min within each individual mixture (12±1.5 or 1.9±0.3 ppm from 5 repeated additions of 10 or 2 CPO units, respectively, see Figure 14, which illustrates studies describing the efficacy of attenuated B. anthracis spores inactivation by CPO-generated oxidizing radicals (TS= thiosulfate). These findings allowed us to test the efficacy of CPO-generated oxidizing radicals (at
5x10 units mixture) to inactivate attenuated B. anthracis spores. Spores were added to 1 ml CPO/NaClO2 mixture and viability was determined after 60 min incubation (see Figure 10). Our data exhibits effective decontamination (at least 6 log reduction) achieved within 60 min at RT (approximately 200C). In addition, NaClO2 by itself (25 mM) did not affect spores viability and the addition of 50 mM sodium thiosulfate to the mixture before spore inoculi prevented spore inactivation (see Figure 14). Based on these results, we will determine the Ct values for CPO-generated oxidizing radicals.
Example 3: Enzyme Based Active Decontamination
This example described the discovery of VX-/Tetriso-degrading enzymes from environmental libraries, and the heterologous expression of novel environmental hits. Hits that have shown up on the VX screen were fully sequenced, and three novel genes were identified, and subcloned into an appropriate E. coli expression plasmid, and host and sequence verified. The clones were His-tagged for culturing and purifying.
Repeated verification of environmental library hits for VX degradation To further substantiate results, 40 environmental library samples were screened.
These 40 lysates were tested for their degradation activity toward VX and Tetriso. The time course of VX degradation and Tetriso degradation by the environmental library lysates was performed with a robotic system.
Figure 15 illustrates data displaying the kinetics of VX (1 μM) degradation by environmental library samples. Time intervals for the kinetics of VX degradation are shown in Figures 15A and 15B. Figures 16A and 16B illustrate data displaying the kinetics of Tetriso (20 μM) degradation by these environmental library samples. Samples for both were taken from the degradation mixture at 7 to 8 specified time intervals up to 85 h (4 d).
The VX degradation library hits detected by this screening process are: SEQ ID NO:76 (encoded by, e.g., SEQ ID NO:75), SEQ ID NO:78 (encoded by, e.g., SEQ ID NO:77), SEQ ID NO:80 (encoded by, e.g., SEQ ID NO:79) (Figure 15A, "plate 1") SEQ ID NO:82 (encoded by, e.g., SEQ ID NO:81), SEQ ID NO:84 (encoded by, e.g., SEQ ID NO:83) and SEQ ID NO:74 (encoded by, e.g., SEQ ID NO:73) (Figure 15B, "plate 2") and to a significantly lesser extent samples SEQ ID NO:86 (encoded by, e.g., SEQ ID NO:85), SEQ ID NO:88 (encoded by, e.g., SEQ ID NO:87) and SEQ ID NO:90 (encoded by, e.g., SEQ ID NO: 89) (Figure 15B, "plate 2") causing only 20 to 25% degradation.
The latter three VX hits: SEQ ID NO:86, encoded by, e.g., SEQ ID NO:85), SEQ ID NO: 88 (encoded by, e.g., SEQ ID NO: 87) and SEQ ID NO:90 (encoded by, e.g., SEQ ID NO: 89), were the only lysates causing significant degradation of Tetriso (Figure 16, plate 2). The other six VX library hits were not detected by Tetriso degradation robotic screening assay. All nine VX hits discovered from the 40 repeated environmental samples were verified by manual kinetic measurement.
Figure 17 illustrates data displaying the kinetics of VX degradation by all nine VX environmental hits compared to a control (cell lysate without any expressed protein), performed manually. The most rapid VX degradation rate was obtained with samples SEQ ID NO:84 (encoded by, e.g., SEQ ID NO:83) > SEQ ID NO:80 (encoded by, e.g., SEQ ID NO:79) > SEQ ID NO:76 (encoded by, e.g., SEQ ID NO:75) with tm values of 12, 19 and 23 h, respectively. The other three hits are SEQ ID NO:78 (encoded by, e.g., SEQ ID NO:77) > SEQ ID NO:82 (encoded by, e.g., SEQ ID NO:81) > SEQ ID NO:74 (encoded by, e.g., SEQ ID NO:73) with t1/2 values 48, 59 and 71 h, respectively. It should be noted that in previous kinetic measurements the sequence of descending order was a bit different, possibly indicating different intrinsic stability of enzymes in crude lysates. Interestingly, it was found that the Tetriso hits SEQ ID NO: 86 (encoded by, e.g., SEQ ID NO:85), SEQ ID NO:88 (encoded by, e.g., SEQ ID NO:87) and SEQ ID NO:90 (encoded by, e.g., SEQ ID NO:89), that also displayed limited degradation activity toward VX, have the same amino acid sequence. The other six VX hits represent two distinct sequences.
Figure imgf000198_0001
* Tetriso library hits
In Figure 17, the time-course of VX enzymatic degradation by environmental library hits was performed manually: 1 μM VX, 250C, with a control cell lysate.
2) Discovery of CPOs from fungal sources Enzymes: The three batches of partially purified fungal heme CPO were assayed on VX. The partially lyophilized samples were reconstituted with water to their original volume by the following dilution factors: 16.6 for the first two samples and 3.33 for the third sample. None of these samples displayed any degradation activity toward VX (10 μM) following 7 d incubation with 0.025 M NaClO2 at 250C. Sequence based discovery: Overlap PCR methodology was used to remove introns from the two full length fungal genes, and the genes are subcloned into an appropriate expression host. Using the same degenerate PCR approach, partial sequences of 10 new peroxidases was found; 4 full length genes were subsequently isolated. Overlap PCR methodology can be used to remove the introns of these genes. 3. Heterologous expression of C. fumago CPO in E. coli, Pichia, Saccharomyces
Nucleic acid (SEQ ID NO:1) encoding C.fumago chloroperoxidase (CPO) (SEQ ID NO: 2) was subcloned into Saccharomyces and can be tested for activity and to see if there is any protein made in the cell using, e.g., using Western blotting. Additionally, the original Pichia constructs were reengineered to add back their native signal sequence (this is an attempt to see if we can get expression in this host). A Western blot of the original constructs made in E. coli and Pichia confirmed that there is no protein being made in these heterologous hosts and this is in keeping with no activity using the MCD assay.
4. Optimization of the heterologous expression of C. fumago in C. heterostrophus
Using the antibody to C.fumago CPO (SEQ ID NO:2) we optimized the detection of the enzyme using western blots in order to detect transformants. It was confirmed, as illustrated in Figure 18 (an anti-C. fumago CPO immunoblot of various samples), 3 to 4 mg/L of chloroperoxidase is being made in C. heterostrophus . This level of expression is not sufficient for GSSM studies, so alternative approaches to improve the CPO expression level will be used.
5. Investigations into CPO inactivation Testing of radical traps: This study was undertaken to determine if the stability of
CPO could be improved by addition of compounds that would absorb potentially inactivating radicals. Nine compounds were tested for sufficient solubility in aqueous systems (to 100 mM) for an initial screening experiment using a minimum amount of inactivating NaClO2 (0.5 mM) that had been previously determined by the proteomics group with CPO (1 μM), including a positive control (no NaClO2 added) and a negative control (NaClO2 only added).
Of the nine compounds, only 4 (POBN, DBNBS, DMPO and sodium salicylate) were sufficiently soluble to carry forward. CPO was incubated for 1 h at RT with each radical trap. Then a time course was taken with aliquots flash frozen in dry ice/acetone at 1', 10', 60' and 24 h after addition OfNaClO2. These samples were lyophilized in preparation for SDS-PAGE analysis. Based on comparison with the positive control (non-inactivated CPO), the ability of radical traps to preserve CPO MW size are ranked:
DBNBS > DMPO > POBN > sodium salicylate Samples will be analyzed by proteomics. Deglycosylation: Conditions for C.fumago chloroperoxidase (CPO) (SEQ ID
NO: 2) were studied under native conditions to explore how well deglycosylated forms of the enzyme perform (to study the enzymatic activity of deglycosylated forms). This was undertaken to evaluate use of heterologous expression methods that cannot glycosylate proteins, e.g., prokaryotic expression systems. Upon SDS-PAGE analysis of time points taken over multiple days a set of successful conditions were identified. This method is tested for reproducibility and can be checked by SDS-PAGE, MS analysis and by the monochlorodimedone assay (chlorination activity assay for CPO).
Exposure of chloroperoxidase to sodium chlorite (proteomics analysis): The current exposure Of NaOCl2 to CPO (25 mM, molar ratio 25,000: 1) was determined. Proteomic Profiling was performed on treated and untreated CPO. The treated CPO was exposed at a ratio of 1 : 1000 OfNaOCl2. Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOFMS) analyses were performed on each sample (of C. fumago chloroperoxidase) and peaks were observed disappearing and appearing between each sample, as illustrated in Figures 19A and 19B. In some cases mass mapping allowed the determination of the sequence for some of the peaks. However, in Figures 19A and 19B the sequence of the peaks which changed in abundance could not be determined.
Electrospray ionization coupled with a quadrupole time of flight mass spectrometry (ESI-QTOFMS) instrument was used to perform tandem mass spectrometry to determine the sequence of peptides from the CPO that were changing in abundance between the untreated and treated samples, as illustrated in Figure 2OA. Peaks which disappeared were selected for tandem mass spectrometry in the untreated sample to determine their sequence, as illustrated in Figure 2OB.
Many peaks are observed to disappear from the untreated to the treated sample in both the MALDI-TOF and ESI-QTOF analysis. If modifications such as oxidation or chlorination were occurring, signals should appear at different masses not disappear completely. It is theorized from previous gel analysis that crosslinking may be occurring. This is a possibility that could explain why many signals in the mass spectrum are absent in the treated sample relative to the untreated sample. Several of the peptides that disappeared when mapped to the protein 3D structure are on the outside of the molecule, as illustrated in Figure 21. Therefore, it is thought that crosslinking may be taking place.
6. Investigation into large-scale decontamination of Tetriso
The amount of chloroperoxidase required to decontaminate 40 mM of the VX analogue Tetriso was investigated. It was found that approximately 1200 U/mL of chloroperoxidase must be present in solution to completely decontaminate Tetriso and prevent the formation of chlorine dioxide, as illustrated in Figure 22. Chlorite consumption, chlorine dioxide formation and residual enzyme activity can be monitored during decontamination. In Figure 22, Tetriso degradation by various concentrations of CPO in 320 mM chlorite and 300 mM sodium phosphate buffer (pH 7.0) is illustrated. 7. Other exemplary enzymes
The invention provides assays for confirming the activity of enzymes of the invention, and determining if a polypeptide has the requisite activity to be within the scope of the claimed invention. One exemplary assay uses a bromide probe for assaying dehalogenase activity (using dibromoethane). A number of buffers and pH will be screened for optimal activity/stability of chloroperoxidase, dehalogenase and DFPase, as illustrated in Figures 23 and 24. Figure 23 illustrates bromide ion formation from dehalogenase-catalyzed hydrolysis of dibromoethane (saturated solution) as a function of enzyme concentration in various buffers. Rates were monitored using a bromide selective electrode. Figure 24 illustrates the rate of fluoride produced by DFPase catalyzed hydrolysis of DFP (3 mM) as a function of DFPase concentration in various buffers. Rates were measured using a fluoride selective electrode. Materials having compatibility with high concentrations of chlorite, chlorine dioxide and the more active versions of the decon formulations of the invention (high concentrations of enzyme and chlorite) can also be used.
Example 4: Enzyme Based Active Decontamination
The invention provides polypeptides having dehalogenase activity, e.g., haloalkane dehalogenase activity, and methods for assaying for dehalogenase activity, e.g., haloalkane dehalogenase activity, and methods for discovering haloalkane dehalogenases. In one aspect, these enzymes can be used to decontaminate sulfur mustard (HD).
I. Enzyme Discovery and Characterization:
This example describes exemplary methods for the discovery, purification and characterization of enzymes to decontaminate Sulfur mustard (HD):
• 13 Haloalkane dehalogenases were grown up and partially purified. These enzymes were tested for activity using a surrogate substrate and 2 of them were tested on sulfur mustard. One enzyme was superior to the other candidates (SEQ ID NO:70, encoded by SEQ ID NO:69) and more than one gram of this enzyme was purified to homogeneity, and used in formulation studies.
Discovery and Isolation of new enzymes for VX degradation
• Screening of environmental DNA libraries: A high-throughput robotic screen was devised and implemented using the surrogate substrate Tetriso. Organophosphorus hydrolase (OPH) was subcloned to serve as a positive control for this assay. In total 12 environmental libraries were screened comprising different ecological niches from around the globe, comprising approximately 1.0 million clones over 3.5 months. In a primary screen 179 clones were identified that appeared to have activity. Each of these clones was grown and lysates were prepared and normalized for cell OD. These samples were direct testing on VX. Nine clones proved to show significant activity on VX (IIBR). These clones were then sequenced and three were identified as unique. All three enzymes belong to the alpha beta hydrolase enzyme superfamily. These genes are currently being subcloned for further characterization. • Isolation of chloroperoxidase from novel fungal isolates: We grew up and assayed more than 500 novel fungal isolates for chloroperoxidase activity, 22 isolates showed activity. Five of these isolates have been grown up and their peroxidase enzymes partially purified. These enzymes are being tested for activity towards VX degradation.
• Sequence based discovery to find novel heme containing chloroperoxidases: Using the known heme chloroperoxidase from C.fumago and homologous genes identified from other public fungal genomes, two degenerate PCR primers were designed and used to pull out similar genes from the same fungal isolates that were screened for chloroperoxidase activity described above. Two full-length sequences were recovered and eight partial sequences were identified.
• Isolation and characterization of Laccases: Laccases have some activity towards VX. 8 laccase enzymes were grown and partially purified, and are being tested for activity on VX. • Vanadium chloroperoxidase: This class of enzymes has previously been shown to be active on VX. Initially this enzyme was partially purified from wildtype cultures of C. inaequalis and the resultant enzyme tested for activity. The gene from Curvularia inaequalis was subcloned into a Cochliobolus heterostrophus fungal host and was over-expressed; the partially purified enzyme was tested for activity. Additionally, a novel vanadium peroxidase was identified from the genome of C. heterostrophus.
Attempts to purify the enzyme from the wildtype organism were not successful due to the co-isolation of polysaccharides with the enzyme.
Heterologous expression of C. fumago CPO
• Filamentous fungal expression: The gene for C. fumago chloroperoxidase was subcloned into a fungal host C. heterostrophus using three different promoters.
Combinations of different promoters and growth media can be used to determine the best conditions for over expression.
• E. coli I Pichia I Saccharomyces: In addition to fungal expression systems, the invention provides E. coli, Pichia and Saccharomyces expression systems. Various constructs plus and minus signal sequences were subcloned into E. coli, Pichia and
Saccharomyces. These transfected cells (including, e.g., cells comprising constructs of the invention, including expressing enzymes of the invention) are being grown and tested for chloroperoxidase activity. • Proteomic analysis of CPO: Using the commercially available C. fumago CPO (SEQ ID NO:2, encoded, e.g., by SEQ ID NO:1) experiments to ascertain why the CPO was losing its activity over time in the presence of VX and NaClO2 were performed. Many different biochemical and proteomic techniques have been used, including several experiments with radical traps. To date we have shown that the enzyme appears to be crosslinking when the enzyme is in the presence OfNaClO2. Oxidation or other modifications at specific amino acids may be occurring on the CPO. Deglycosylating the enzyme may aid subsequent proteomic analysis; and, testing various digest conditions may also aid subsequent proteomic analysis - for best coverage of the enzyme by mass spectrometry.
II. Surrogate Agent Decontamination and Formulations: DFPase and G agent degradation
• In order for DFPase to be used in combination with chloroperoxidase, it must maintain activity in the presence of sodium chlorite and chlorine dioxide. When DFPase (2.5 mg/mL) is incubated with sodium chlorite (O - 100 mM) or chlorine dioxide (0 - 100 ppm) significant activity (>30%) is retained after 30 min of incubation.
• Chlorine dioxide does not degrade DFP. DFPase does degrade 2.5 mM DFP in the presence of 100 ppm Of ClO2; however, increased concentrations of DFPase are required to make up for the reduced activity.
• Since two moles of acid are produced for every mole of agent hydrolyzed, buffer is necessary to guarantee complete degradation of significant agent.
• DFPase is effective in the degradation of 1 wt% DFP as long as significant buffer is present to prevent a decrease in pH. 200 mM ammonium carbonate buffer was effective in preventing pH change and allowed for complete degradation of 1 wt%
DFP by 8 U/ml of DFPase in 20 min.
• DFPase was effective in decontaminating 1 wt% DFP in the presence of up to 100 mM sodium chlorite. The addition of 50 U/ml of chloroperoxidase with 100 mM sodium chlorite did not prevent complete DFP degradation. Chloroperoxidase and Oxidative decontamination
• Chlorine dioxide deactivates both chloroperoxidase and horseradish peroxidase and that the deactivation is associated with a decrease in the Soret absorption band for the enzymes. • 0.5 niM tetriso is effectively degraded by 50 U/ml of chloroperoxidase with 25 mM sodium chlorite at pH 7.0. Under these conditions, only 14 ppm of ClO2 was detected in solution. In order to obtain similar degradation using chlorine dioxide, an excess of ClO2 is required.
• Studies showed that 2-CEES are quickly degraded to its sulfoxide by chloroperoxidase with sodium chlorite.
• 109 B. subtilis vegetative cells are completely deactivated after exposure to 50 U/ml of horseradish peroxidase or 50 U/ml chloroperoxidase with sodium chlorite for 30 min at pH 7.5.
• B. subtilis spores are deactivated after exposure to chloroperoxidase and horseradish peroxidase with 25 mM sodium chlorite at pH 7.5; however, repeated addition of enzyme is required for complete inactivation.
• We have measured the stability of chloroperoxidase and horseradish peroxidase after addition of 25 mM sodium chlorite with and without 0.5 mM tetriso. The half-life of 50 U/ml enzyme under these conditions is about 3 min for chloroperoxidase and about 1 min for horseradish peroxidase.
• A number of solution additives (superoxide dismutase, catalase, PEI, guiaicol, ascorbic acid, ABTS, mannitol and DMSO) were investigated for their ability to improve chloroperoxidase stability during decontamination. None of the stabilizers tested were effective in improving enzyme stability. • Controlled addition of chlorite was investigated as a technique to improve chloroperoxidase stability and increase tetriso decontamination. Although the half- life of chloroperoxidase was improved, the rate of tetriso degradation was decreased and no significant improvement in tetriso turnover was observed.
• Using 160 mM chlorite and 960 U/mL chloroperoxidase (in 300 mM phosphate buffer, pH 7) we were able to decontaminate nearly 25 mM tetriso in 10 min.
• Addition of 50 U/ml of chloroperoxidase to solution of chlorine dioxide (approximately 60 ppm) causes a large decrease in the chlorine dioxide (nearly 80%). Addition of an equivalent weight of BSA causes a negligible change in chlorine dioxide concentration. Corrosion Studies
Corrosion studies comparing chlorine dioxide (340 or 800 ppm) with the enzymatic decontamination (decon) formulation (DFPase, Chloroperoxidase, 25 niM sodium chlorite) showed the enzymatic decontaminant to be less corrosive to metals. Figure 25 illustrates results of these studies - describing degradation of 1 wt% DFP by DFPase (8 U/mL) in various concentrations of ammonium carbonate buffer (50-300 mM).
Chloroperoxidase and oxidative decontamination Figure 26 illustrates studies regarding chloroperoxidase and oxidative decontamination - in this study there was a change in the Soret absorption band for chloroperoxidase upon addition of chlorine dioxide. In Figure 26, the Soret band (398 nm) disappears upon additon of chlorine dioxide. Loss of enzyme activity also corresponded with loss of Soret band. Figure 27 illustrates studies demonstrating degradation of tetriso by chlorine dioxide and sodium chlorite, in 50 mM phosphate buffer, pH 7.0.than 50% of the tetriso after 20 minutes. Figure 28 illustrates studies demonstrating degradation of tetriso by chloroperoxidase/ NaClO2/ ClO2 at pH 7.0 in 50 mM phosphate buffer. Figure 29 illustrates studies demonstrating inactivation of B. subtilis var Niger by sodium chlorite and bioxidation using chloroperoxidase or horseradish peroxidase after 1 hr of exposure. All experiments were in 50 mM phosphate buffer, pH 7.5. Figure 30 illustrates studies demonstrating inactivation of B. subtilis var Niger spores by sodium chlorite and bioxidation using chloroperoxidase or horseradish peroxidase after 1 hr of exposure. Enzymes were added as 50 units in a single shot (1X50) or as 5 additions (5X10). All experiments were in 50 mM phosphate buffer, pH 7.5. Figure 31 illustrates studies demonstrating the stability of chloroperoxidase in buffer, in 25 mM NaClO2, in NaClO2 with 0.5 M tetriso, and in 0.25 mM ClO2. Samples were removed periodically and assayed using MCD. Figure 32 illustrates studies demonstrating the stability of horseradish peroxidase in buffer, in 25 mM NaClO2, in NaClO2 with 0.5 M tetriso, and in 0.25 mM ClO2. Samples were removed periodically and assayed using Guiaicol and H2O2. Figure 33 illustrates studies demonstrating Tetriso degradation by 480 u/ml of CPO in 300 mM phosphate buffer (pH 7.0) with 80 mM NaClO2. No chlorite was available at the end of the run and the pH changed by only 0.15 units. For control, no pH change, 10 mM chlorite gone after one hour. Figure 34 illustrates studies demonstrating Tetriso degradation by 300 mM Phosphate buffer (pH 7.0) with 160 mM NaClO2 (no enzyme). Approximately 60 mM NaClO2 disappeared after one hour and pH change approximately 0.3. Figure 35 illustrates studies demonstrating Tetriso degradation by 960 u/ml of CPO 300 mM phosphate buffer (pH 7.0) with 160 mM NaClO2. pH change at end of one hour was approximately 0.3 to 0.4 and negligible chlorite was present at the end of one hour. Figure 36 illustrates studies demonstrating a decrease in ClO2 absorbance upon the addition of 50 u/ml of CPO. Addition of BSA caused negligible change in absorbance. Figure 37 illustrates studies demonstrating the kinetics Of ClO2 degradation upon addition of CPO (50 u/ml) or an equivalent weight of BSA. Corrosion Studies on Metals
The invention provides formulations (e.g., mixed solutions) comprising haloperoxidase enzymes of the invention (e.g., chloroperoxidases) and a chlorite component, e.g., sodium chlorite, or equivalent (e.g., sodium iodite would be an equivalent). In one aspect, these formulations are liquids that are applied as decontamination either before, during and/or after a toxic exposure event. In another aspect, these formulations are applied as coatings to, or can be sprayed onto, a surface, e.g., to cloth, textiles, metals (e.g., steel materials), elastomers, plastics, alloys and the like. The enzyme component and the chlorite component, e.g., sodium chlorite, or equivalent, component can be applied together as one formulation or as a mixed co- application, or, they can be applied separately (in any order). Chlorine dioxide is generated by this decontamination reaction. These studies (described in detail, below) using steel-based materials clearly demonstrate that the amounts of chlorine dioxide generating by practicing this aspect of the invention is non-corrosive to steel materials to which they can be applied. While the invention is not limited by any particular mechanism of action, the hypothesized mechanism of action of the decontamination action of chloroperoxidase is to convert sodium chlorite (or equivalent) to chlorine dioxide (or equivalent) - and it is the chlorine dioxide (or equivalent) which the effective decontamination agent; noting, the enzyme can make many oxidizing species, but these have not been characterized. In some conventional decontamination protocols chlorine dioxide alone is used to decontaminate; and in these conventional applications chlorine dioxide is generally used at relatively high concentrations - approximately 300 to 787 ppm - that are corrosive to metal and other surfaces. In fact, even the relatively high concentration of 300 ppm does not degrade VX simulant effectively. When chlorine dioxide is generated enzymatically by practicing this invention, the concentrations of chlorine dioxide needed to effectively decontaminate can be much lower, for example, in one aspect, approximately 100 ppm. Thus, in these studies we are comparing low concentrations of chlorine dioxide (to simulate enzyme-generated conditions) to high concentrations (as used in conventional decontamination), particularly their corrosive effect on various metals.
Figures 38 and 52 illustrate studies (described in detail, below) demonstrating the non-corrosive effect on steels when practicing (using) the decon compositions and methods of the invention comprising haloperoxidase enzymes of the invention and a chlorite component. For example, Figure 38 illustrates two photos of blue-tempered spring steel by enzyme-based decontamination solution (left panel) and 340 ppm chlorine dioxide (right panel); this study demonstrates that there is no corrosion with this exemplary enzyme-based decontamination solution, (see also discussion on Figure 25, above)
Figure 52 illustrates four photos of 110 carbon steel exposed to levels of chlorine dioxide "normally" generated when practicing and using these haloperoxidase/ chlorite component decontamination (decon) formulations of the invention. This study also demonstrates the non-corrosive effect of these compositions of the invention. As shown in Figure 52, neither the before (upper left photo) nor the after (upper right photo) of the carbon steel exposured to a concentration of chlorine dioxide equivalent to that amount generated by an exemplary decontamination (decon) formulation of the invention showed any significant (i.e., any visual) signs of corrosion. In contrast, significant (i.e., visually detectable) corrosion was seen in the steel sample exposed to a concentration of chlorine dioxide three times higher than that generated by using this exemplary decontamination (decon) formulation of the invention (used to generate the upper two photos) - before exposure is the lower left photo, and after exposure is the lower right photo.
In addition to the illustrated steel material, elastomers, metals, and alloys passed submersion testing with both decontamination (decon) formulations of the invention, and exposure to concentrations of chlorine dioxide the exemplary decontamination (decon) formulations of the invention generate when practiced. In contrast, some metals failed when exposed to concentrations of oxidizing agent lower than or equivalent to those used in chlorine dioxide sprays.
The invention includes formulations of haloperoxidase enzymes of the invention (e.g., chloroperoxidases) and a chlorite component, e.g., sodium chlorite, or equivalent (e.g., sodium iodite would be an equivalent) for use as primers and coatings, on or in textiles and clothing, on or in plastics, and the like.
Protocol for corrosivity testing of metals and alloys (for data in Figures 38 and 52)
Using the following types of metals and alloys: 304, 316, and 321 stainless steel; 110 carbon steel; Nickel steel; Blue spring steel; Copper; Hard- and soft-tempered aluminum. The corrosivity of different solutions (comparison of 100, 300, and 787 ppm of chlorine dioxide) was tested, as described, below. The solutions that we used were all dilutions of chlorine dioxide in deionized water.
100 ppm chlorine dioxide This is the concentration of chlorine dioxide generated during the degradation of
VX-simulant via chloroperoxidase/sodium chlorite decontamination. When VX simulant is not present, approximately 50 ppm of chlorine dioxide is generated.
300 ppm chlorine dioxide, 787 ppm chlorine dioxide
These concentrations are approximately equivalent to or less than concentrations of chlorine dioxide used in decontaminations. We found that 300 ppm chlorine dioxide (synthesized in our laboratory) does not degrade 1% w/v VX simulant in 30 minutes.
The testing was performed using ASTM (American Society for Testing and Materials) method G31-72 (1995), Laboratory Immersion Corrosion Testing of Metals. The other method that was used was ASTM F502-93 to visually observe changes to the metals and alloys.
The metals and alloys were cut into uniform coupons, and their area was checked using a caliper. The coupons were washed and dried. They were weighed in triplicate and the average was calculated. Each coupon was placed in a 100 mL wide-mouth jar and immersed in the testing solution. The jars were sealed, and the samples were left in the solution for approximately 24 hours. The coupons were removed from the solution, rinsed with water, and air dried. The appearance of corrosion was recorded using photographs. Corrosion products were removed with a soft brush. The coupons were again weighed in triplicate and the average mass was calculated.
In order to determine corrosion rate, the loss of mass, time of exposure (in hours), area, and density of the coupons were used to determine the corrosion rate (in mils per year). The following equation was used:
Corrosion rate = (K x W)/(A x T x D)
Where K is a constant (in mils per year = 3.45 x 10 )
A= the area in cm2
T= time of exposure in hours
W= the mass loss in grams (to nearest mg)
D= the density (in g/cm3)
This assumes that the rate of corrosion is uniform. The corrosion was classified using the National Association of Corrosion Engineers (NACE) standard TMO 169-2000.
The tables, below, present a comparison of 100, 300, and 787 ppm of chlorine dioxide solutions. The data show that 110 Carbon steel is strongly corroded by 787 ppm, less so by 300 ppm, and least so by 100 ppm. The results for other metals, say Blue Spring Steel, are similar.
100 ppm CIO2 - Shorter Immersion
Figure imgf000209_0002
Time (hours) Constant (mpy) 18.75 3.45E+06
Thickness (cm)
0.00508
Figure imgf000209_0001
Figure imgf000209_0003
Figure imgf000210_0002
Constant
Time (hours) (mpy) 19.25 3.45E+06
Figure imgf000210_0003
Constant
Time (hours) (mpy)
16.00 3.45E+06
Figure imgf000210_0001
The ratio of chloroperoxidase to sodium chlorite is important, in some cases because of the amount of VX-simulant we can decontaminate, but mostly because of the amount of chlorine dioxide we generate. In practicing some aspects of this invention is it desirable to design ratios of an enzyme of the invention and a substrate (e.g., a chlorite component, as sodium chlorite) that give complete decontamination while generating 100 ppm or less of chlorine dioxide. In one aspect, the chlorine dioxide is generated solely for the purpose of decontaminating a biological agent, such as a spore, e.g., an anthrax, or Bacillus anthracis, spore. Thus, in at least this aspect (decontaminating biological agents, such as spores), it is not needed to generate as much chlorine dioxide as in alternative aspects of the invention for decontaminating V agents, e.g., VX, etc., as described herein. While the invention is not limited by any particular mechanism of action, in one aspect, chlorine dioxide itself does not decontaminate the VX simulant. We have done experiments with synthesized chlorine dioxide (as opposed to enzyme-generated) and found that even 300 ppm does not decontaminate 1% w/v VX simulant in 30 minutes. In fact, there is no noticeable degradation. We believe that what is actually decontaminating the VX simulant are short-lived oxidizing species. We have not isolated or determined which species are responsible for the decontamination, as it is sufficient that studies have clearly demonstrated that the enzymes, formulations and methods of the invention can generate such effectively decontaminating (if not short-lived?) oxidizing species.
III. Live agent studies:
Evaluation of enzymes for decontamination of Sulfur Mustard (HD): The following example described exemplary assays that can be used to determine if a polypeptide has requisite activity to be within the scope of the invention. The following example also describes the identification and characterization of exemplary enzymes of the invention.
• 14 new clones of dehalogenase (DHG) were tested for the degradation kinetics of sulfur mustard (HD) and sulfur mustard-sulfoxide (HD-SO). The DHG clone encoding the exemplary dehalogenase (DHG) SEQ ID NO:70 (encoded, e.g., by SEQ ID NO:69) displayed a remarkable activity toward sulfur mustard (HD) degradation. The i\a for sulfur mustard (HD) (50 μM) degradation by the exemplary SEQ ID NO:70 (encoded, e.g., by SEQ ID NO:69) is less than 1 min (250C, pH 7.5), as illustrated by Figure 39, showing the degradation of sulfur mustard (HD) by the dehalogenase (DHG) SEQ ID NO:70 (BD 2036 in figure) (encoded, e.g., by SEQ ID NO:69) and SEQ ID NO:92 (BD 2037 in figure) (encoded, e.g., by SEQ ID NO:91) (sulfur mustard (HD) 50 μM, DHG 2 mg/ml, 50 mM Phosphate, pH 7.5, 3O0C, n=3 per each time interval:
Figure imgf000211_0001
This degradation rate is at least 10-fold more rapid than the rate of sulfur mustard (HD) hydrolysis in buffer and depends on dehalogenase (DHG) (Figure 40) and sulfur mustard (HD) concentration (Figure 41). Figure 40 illustrates data showing the residual percent sulfur mustard (HD) following enzymatic degradation by the exemplary SEQ ID NO: 70 at various DHG concentrations (HD 50 μM, SEQ ID NO:70, 2 min, phosphate 50 mM, pH 7.5, 3O0C, the table above contains duplicate data sets, done in triplicates per each DHG concentration):
Figure imgf000212_0001
Figure 41 illustrates data showing the residual percent sulfur mustard (HD) following enzymatic degradation by the exemplary SEQ ID NO:70 at various HD concentrations (DHG 2 mg/ml, 2 min, Phosphate pH 7.5, 3O0C):
Figure imgf000212_0002
Significant sulfur mustard (HD) enzymatic degradation (45% to 50%) with the exemplary SEQ ID NO: 70 was obtained at 1 to 3 min even at low temperature (0-20C) indicating low activation energy for enzymatic de-chlorination of HD. Sulfur mustard (HD) degradation was obtained even after re-use of the same SEQ ID NO:70 batch with another portion of HD (50 μM) (Figure 39) indicating resistance of DHG toward HD-induced inactivation.
The effect of propylene glycol (1,2-dihydroxypropane, PG) and polyethyleneglycol-400 (PEG-400) on DHG-catalyzed HD degradation (2 minute incubation) was examined. PEG-400 at 2% or 5% does not modify the level of sulfur mustard (HD) enzymatic degradation compared to buffer: 74%, 76% and 72% degradation at 0%, 2% and 5% PEG, respectively, as illustrated in Figure 42. 10% PEG-400 reduces by about 10% the sulfur mustard (HD) enzymatic degradation level to 65%. These results indicate compatibility of PEG-400 for dissolution and decontamination of HD especially at higher concentrations.
Figure 42 illustrates data showing the degradation of sulfur mustard (HD by the exemplary SEQ ID NO:70 with polyethyleneglycol-400 (PEG-400) at a dehalogenase (DHG) concentration of 2 mg/ml, at 2 min, and buffer comprising phosphate 50 niM, pH 7.5, 25°C.
Co-Enzymatic Degradation of HD: Degradation of sulfur mustard (HD) (50 μM, 250C, pH 7.5) by the exemplary dehalogenase (DHG) SEQ ID NO:70 (2 mg/ml) was examined in the absence or presence of CPO/NaClO2 (note: this is an exemplary enzyme/substrate mixture for HD and VX degradation). Figure 43 illustrates data displaying that CPCVNaClO2 causes nearly complete (>99%) degradation of sulfur mustard (HD) within 1 min. However, CPO/NaClO2 in the presence of DHG caused lower HD degradation (residual HD 18%). Figure 43 illustrates data displaying the time-dependence of HD degradation by the exemplary SEQ ID NO:70 and CPO+ NaClO2 at HD 50 μM, DHG 2 mg/ml, CPO 10 U/ml (x5), NaClO2 0.5 M, Phosphate pH 7.5, 250C.
Furthermore, at 3 min there is a complete degradation of HD either with CPO/NaClθ2 or in the presence of CPO/NaClO2 combined with dehalogenase (DHG), as illustrated in Figure 44. Comparison of HD degradation at fixed time interval (2 min) corroborates our kinetic data. Figure 44 displays the residual HD levels following degradation by DHG, CPO/NaClO2, NaClO2 and combined mixture of DHG+CP0/NaClO2. CPO/NaClO2 causes complete degradation of HD within 2 min. In the presence of either DHG alone or DHG combined with
CPO/NaClO2 degradation of HD within 2 min is 90 and 80% respectively (Figure 44). In Figure 44, the degradation of HD by the exemplary SEQ ID NO:70 and CPO+NaClO2 was within 2 min, at HD 50 μM, DHG 2 mg/ml, CPO 10 U/ml (x5), NaClO2 0.5 M, Phosphate pH 7.5, 250C. The exemplary dehalogenase (DHG) SEQ ID NO:70 was also the most active hydrolyzing enzyme for degradation of HD-SO (the oxidation product of HD using CPO). Figure 45 displays the residual level of HD-SO obtained at 40, 60 and 180 min in the absence or presence of the exemplary DHG SEQ ID NO:70, 2 mg/ml. The residual HD-SO concentration in the presence of DHG is 8% compared to 71 -77% residual HD-SO incubated in buffer within 40 min (Figure 45). Thus, DHG is essential for HD decontamination in addition to CPO since it detoxifies completely HD-SO that is still relatively toxic. Figure 45 illustrates the time course of_enzymatic degradation of HD-SO by the exemplary SEQ ID NO:70 at HD-SO 100 μM, DHG 2 mg/ml, phosphate pH 7.5, 250C; extraction of HD-SO from buffer with ethyl acetate in the presence of 1 M NaCl provides 90% recovery of HD-SO.
Identification and stability of HD enzymatic degradation products Thiodiglycol (TDG) was identified by GC/MS as the main dehalogenase
(DHG) hydrolysis product of sulfur mustard (HD), indicating practically complete detoxification of HD. The enzymatic oxidation products of HD identified by GC/MS (HD-sulfoxide and HD-sulfone) were synthesized and served for studying their hydrolytic stability and as reference compounds for identification of enzymatic peroxidation products of HD.
The hydrolysis products of sulfur mustard (HD) identified by 1H-NMR are: monochloro-monohydroxy-HD and thiodiglycol (TDG), for HD-SO: monochloro-ethyl vinyl sulfoxide and for HD-SO2: monochloro-ethyl vinyl sulfone and divinyl sulfone. Half-life time (ti/2) values evaluated by 1H-NMR for HD, HD-SO2 and HD-SO hydrolysis are: 10, 250 min and 1,222 hr, respectively (50 mM phosphate, pH 7.5, 250C).
Development of assay conditions for VX degrading enzymes using high throughput screening (HTS) robotic system: Validation of the robotic system diagnostic function was performed by simulated degradation of VX in 96-well plates array. The kinetics for AChE inhibition by VX (10"9-10~8 M) was measured using the robotic system coupled to on-line 96-well plate optical reader. The kinetic results conformed well to previous data obtained manually.
Short (15 sec- 10 min) and long-term (10-120 min) degradation kinetics experiments were conducted with VX using C. fumago chloroperoxidase (CPO) (SEQ ID NO:2) (NaClO2 pH 7.5) for testing the software control and hardware interactive performance. The results conform quite well to those obtained manually.
Since high throughput screening (HTS) of DNA libraries was conducted using Tetriso as a non-toxic VX surrogate, it was important to examine its degradation rate compared to VX. The enzymatic degradation activity of C. fumago CPO toward Tetriso compared to VX was measured. The rate of VX degradation by CPO was very similar to that of Tetriso (kobs=0.15 and 0.14 min"1, respectively, using 0.1 mM phosphorothiolates, CPO 40 U/ml, NaClO2 0.025 M, pH 7.5, 250C. Therefore, Tetriso could in principle be used as VX surrogate in searching for new peroxidative enzymes.
VX degradation kinetics catalyzed by 0.1, 0.5 and 1 U/ml CPO was measured with the robotic system at two VX concentrations: 1 and 10 μM (Fig. 8). Prior to these experiments, the kinetics of VX degradation was measured at various NaClO2 concentrations (0.01-0.1 M) with fixed CPO activity. These experiments enabled the selection of suitable NaClO2 concentrations for low CPO activity. Figure 46 illustrates the time-course of VX degradation by C.fumago chloroperoxidase (CPO) (SEQ ID NO:2) at low activity level, Figure 46A=VX 10 μM, NaClO2 0.03 M; Figure 46B= VX 1 μM, NaClO2 0.02 M, Phosphate 5OmM, pH 7.5, 250C:
Figure imgf000215_0001
Evaluation of new enzymes for VX decontamination
Native C. inaequalis VaCPO: Addition of a single portion of 80 μl VaCPO (0.2 mg/ml) provided similar degradation rate (t]/2 = 13 min) to that obtained with 5x20 μl VaCPO. (t1/2=15 min), (NaCl 0.5M, UPER 0.5 mM, pH 2.75, 50 mM tartarate). No VX degradation was obtained with VaCPO using NaClO2 as co- substrate (0.01-0.1 M) at pH 7.5.
Recombinant C. inaequalis VaCPO: A sample of recombinant C. inaequalis VaCPO expressed in C. heterostrophus containing total activity of 16.2 U as measured by monochlorodemedon (MDM) chlorination assay. Data showed that there is practically no degradation of either VX or Tetriso in the presence of recombinant C, inaequalis VaCPO.
Recombinant fungal laccase: We have previously noted (Amitai et al., FEBS Lett, 1998) that Pleurotus ostreatus laccases could degrade VX in the presence of ABTS as co-substrate using ambient oxygen dissolved in buffer (phosphate pH 7.4). Two samples of recombinant fungal laccase (phenol oxidase) were used. Degradation rates obtained with laccase are significantly slower compared to degradation rates obtained with chloroperoxidase. Environmental library fluorescent hits: 179 enzymes derived from environmental library hits were selected from a larger collection (20,000 clones) on the basis of degradation activity toward lOμM Tetriso (VX surrogate). Overall, 10 host controls and 179 fluorescent hits were screened for VX degradation activity by the robotic system at IIBR. All samples were monitored daily for 6 to 8 days.
Figure 47 describes the kinetics of VX degradation measured in a representative one 96-well plate (out of eight) containing 24 lysate samples (out of 179 samples) at specified time intervals. Out of 179 environmental samples the following nine hits displayed significant degradation activity toward VX (all verified by a manual kinetic experiment): SEQ ID NO:74 (encoded by, e.g., SEQ ID NO:73); SEQ ID NO:76 (encoded by, e.g., SEQ ID NO:75); SEQ ID NO:78 (encoded by, e.g., SEQ ID NO: 77); SEQ ID NO:80 (encoded by, e.g., SEQ ID NO:79); SEQ ID NO:82 (encoded by, e.g., SEQ ID NO:81); SEQ ID NO:84 (encoded by, e.g., SEQ ID NO: 83); SEQ ID NO: 86 (encoded by, e.g., SEQ ID NO:85); SEQ ID NO:88 (encoded by, e.g., SEQ ID NO:87) and SEQ ID NO:90 (encoded by, e.g., SEQ ID NO:89). Figure 47 illustrates the time-course of Tetriso degradation by environmental library lysates at 20 μM Tetriso, 250C, measured by the high throughput HTS robotic system, one representative 96-well plate out of eight.
All 179 lysates were also tested toward Tetriso degradation using the robotic system. Figure 48 illustrates data showing that there were only three Tetriso hits discovered by the robotic screening assay, these are samples number SEQ ID NO:86 (encoded by, e.g., SEQ ID NO:85), SEQ ID NO:88 (encoded by, e.g., SEQ ID NO:87) and SEQ ID NO:90 (encoded by, e.g., SEQ ID NO:89). The other six VX hits were not detected by the Tetriso assay. These results indicate that Tetriso could not always serve as a VX surrogate (especially for hydrolases). Summary of all VX and Tetriso degradation kinetics for all library hits is depicted in Figure 49 and Figure 50, respectively.
To summarize VX and Tetriso degradation by environmental hits: as illustrated in Figure 49A: Time-Course of Enzymatic Degradation of VX by Lysates (1 μM VX, 250C):
Figure imgf000216_0001
Figure imgf000217_0001
Figure 49B illustrates the time-course of enzymatic degradation of Tetriso by lysates (20 μM Tetriso, 250C).
Evaluation of hydrolases for G-Agent decontamination
DFPase Activity toward DFP and GF: The activity of recombinant Loligo vulgaris DFPase (rec. squid DFPase) was evaluated toward DFP and Cyclohexylsarin (GF). Two different batches of DFP were evaluated at equivalent nominal activities: 1. DFPase (Biocatalytics, USA); 2. DFPase (Roche Diagnostics). These enzymes display similar activity toward GF degradation. The Roche Diagnostics DFPase seems to be more active toward DFP, see Table, below. The rate of DFP and GF degradation by both DFPase batches is inversely proportional to DFP and GF concentrations, summarized in this Table:
Figure imgf000217_0002
(1)A = DFPase from Biocatalytics, USA, G = DFPase from MOD, Germany
It is pertinent to note that the rate of GF degradation at 0.1 mM by DFPase is slower by about two of orders of magnitude compared with DFP degradation rate, see Table above.
Degradation of VX by CPO and DFP enzymatic hydrolysis by DFPase in concentrated buffers: DFPase-induced enzymatic hydrolysis of DFP at relatively high concentration (50 mM) proceeds to completion in 0.2 M ammonium carbonate (AC) buffer at pH=8. Therefore, it was important to evaluate the kinetics of VX degradation by CPO using 0.2 M AC. Figure 51 summarizes data indicating that VX oxidation by CPO/NaClθ2 (shown) as well as GF (cyclohexyl sarin) and DFP hydrolysis by DFPase (not shown) in 0.2 M phosphate proceed to completion at a relatively rapid rate, whereas VX degradation in 0.2 M AC is slow and incomplete (even after repeated addition of CPO) (see Figure 51). Therefore, concentrated phosphate buffer (0.2 M) is more compatible with both enzymes (i.e. DFPase and CPO) than AC buffer.
Example 5: Methods for discovery of organophosphoesterases of the invention
The invention provides organophosphoesterases which rapidly detoxify a pesticide, herbicide and/or insecticide by hydrolyzing P-S or P-F bonds. The following example describes assays that can be used to determine of a polypeptide has organophosphoesterase activity and can, e.g., detoxify a pesticide, herbicide and/or insecticide, and be within the scope of the invention.
Pre-assay: Bioassay trays containing LB/Kan50 were spread with excised amplified libraries and grown overnight to an appropriate density for the CPS group. Enough trays were generated to pick colonies to fill 100 pre-barcode labeled black 384- well plates per day, at a rate of one library per week. The plates were incubated at 370C overnight. The next day these "mother" plates were then pintool replicated into storage "daughter" plates (again pre-barcode labeled and charged with 50 ul LB/Kan50). Tetriso (10 ul of 5OuM solution in Cel-Lytic; final concentration approximately 10 uM) was dispensed into each mother plate. Every tenth mother plate contained a host only positive control in the A1-A2-B1-B2 corner containing no toxin, to serve as a reference fluorescent signal for each run of plates. These plates were covered with robolids (Corning, 3089), bagged (Ziploc) and placed in a humidified incubator at 37C for 6 days. The daughter storage plates were treated with glycerol (10 ul of 50% solution) and stored at -2O0C. Assay: Plates were assayed in batches of 100 by robot daily. Plates were loaded onto the robot carousel, where butytrylcholine esterase (lOul of a 20 U/ml solution) addition was followed by shaking to permit inactivation of the cholinesterase by any remaining un-degraded Tetriso. Then addition of resorufin butyrate (10 ul of a 0.25mM solution) was followed by shaking and endpoint visualization at X^x 560nm; λem 610nm. To simplify downstream waste processing, the plates were also treated with bleach (10 ul
. after visualization. Data analysis: All data were examined using an enzyme kinetics analysis software program. All signals greater than one sigma above background were carried forward to breakout.
Breakout: All hits were streaked out, and individual colonies were manually re- inoculated into black 384-well plates in triplicate to confirm reproducibility by robot assay. Hits that reproduced signal by this assay were lyophilized as crude lysates for later testing on the live agent VX.
Example 6: Decontamination of attenuated B. anthracis spores.
The invention provides compositions (e.g., enzymes and formulations) and methods for decontaminating, detoxifying and/or neutralizing biological agents, e.g., bacillus spores, such as B. anthracis spores. This example demonstrates the effectiveness of the compositions and methods of the invention, in particular, the exemplary chloroperoxidase (CPO) of the invention SEQ ID NO: 2, in decontaminating bacterial spores using attenuated B. anthracis spores as an exemplary model system, a sporocidal assay.
A primary standard assay for the evaluation of spore decontamination by chloroperoxidase (CPO) (SEQ ID NO:2) in a volume of 1 ml had been defined. This assay is based on a pre-made mixture, for the preparation of the CPO-generated oxidizing radicals, into which the spores are added. We determined that the most effective composition of the pre-made mixture is: 5OmM phosphate buffer, 10OmM NaClO2 and CPO 10 units/ml. The feasibility of using 100 mM phosphate buffer in this sporocidal assay was studied; efficacy tests of spore decontamination with a CPO enzyme can be done with this sporocidal assay.
Since 200 mM phosphate buffer was found to be inadequate for use in CPO/NaClO2 mixtures for B. anthracis decontamination, the feasibility of using an intermediate phosphate buffer concentration (10OmM) was tested. In order to optimize the conditions for formation of oxidizing radicals in 100 mM phosphate buffer, different CPO amounts (10 to 500 u/ml) were tested in combination with 100 mM or 50 mM NaClO2. The results demonstrate that all mixture containing 100 mM phosphate buffer yielded lower amounts of CPO-generated oxidizing radicals when compared to their parallel 5OmM controls.
NaClO2 concentrations were found to play a pivotal role in the formation of oxidizing radicals in mixtures containing 10 OmM phosphate buffer. Mixtures containing 50 mM NaClO2 and 10 units/ml CPO yielded very low levels of CPO-generated oxidizing radicals (3 ppm to 3.5 ppm). Higher CPO concentrations (50 to 250 units/ml) only slightly enhanced the level of CPO-generated oxidizing radicals (3 to 7 ppm). When 10OmM phosphate buffer was combined with 10OmM NaClO2, oxidizing radicals formation was improved (8.7 to 10 ppm) but still remain significantly lower then the 5OmM phosphate buffer parallel controls (12-13ppm). In addition, the CPO-generated oxidizing radicals level (9 to 11.5 ppm) was found to be basically unaffected by the CPO concentrations (10-500 units/ml) in mixtures containing 10OmM phosphate buffer and 10OmM NaClO2. Although the efficiency of oxidizing radicals formation by CPO in 100 mM phosphate buffer was lower relatively to mixtures containing 50 mM phosphate, we decided to test the efficacy of spore decontamination by mixtures containing 100 mM phosphate buffer, 100 mM NaClO2 and different CPO concentration (10, 50, 100, 250 or 500 units/ml). Attenuated B. anthracis spores were added to each mixture (containing 9- 13 ppm of CPO-generated oxidizing radicals) and were incubated for 20-40 min in order to obtain Ct values between 200-800 (min x mg/ml). The sporocidal activity of the CPO- generated oxidizing radicals was significantly impaired at Ct values between 400-800 (min x mg/ml), which previously shown to be effective of decontaminating 6 logs in the standard sporocidal assay with 5OmM phosphate buffer. In addition, CPO-generated oxidizing radicals reproduced in mixtures containing 10OmM phosphate seem to have lost their sporocidal activity as CPO concentration were increased above 50 units/ml at the same Ct levels.
Our observation that similar Ct values result in striking differences in spore decontamination efficacy when 50 mM or 100 mM phosphate buffer mixtures were used, led us to investigate whether it is due to a weaker stability of the radicals in stronger buffer environment. For that purpose, stocks of CPO-generated oxidizing radicals were formed in mixtures containing 50 mM phosphate buffer, 100 mM NaClO2 and 10 CPO units/ml, were then diluted into 50 or 150 mM phosphate buffer (1:1, v/v) in order to achieve a final phosphate concentrations of 50 and 10OmM, respectively. Attenuated B. anthracis spores were added to each mixture and incubated for 60 min to obtain Ct values of 1320-1740 for the stock 5OmM solution, and 570-660 or 515-700 for final phosphate concentrations of 50 and 100 mM respectively. The results demonstrate complete decontamination of the inoculated spores (at least 6 log reduction in viability) in both treatments (final phosphate concentrations of 50 and 100 mM) as well as in the original stock solution. These findings suggest that 100 mM phosphate buffer does not interfere with the sporocidal activity of the CPO-generated oxidizing radicals after their formation in 50 mM phosphate buffer. Thus, the inability of oxidizing radicals prepared in 100 mM phosphate buffer to kill spores might be linked to hampered radicals formation at higher phosphate concentrations.
The exemplary CPO of the invention (SEQ ID NO:2) was prepared in solutions at 130 units/ml; and preliminary compatibility tests were performed. CPO was diluted to a stock solution of 200 units/ml, which was used in a series of 1 ml volume standard sporocidal assays (50 mM phosphate buffer, 100 mM NaClO2 and 10 units/ml CPO). As control, a commercial CPO (Sigma) was used. Both enzymes produced similar amounts of CPO-generated radicals (10-11 ppm), and after 60 min of incubation (Ct = 600-660) full decontamination of the inoculated spores achieved (at least 6 log reduction in viability). These experiments imply that the CPO of the invention is compatible with all reagents and can be used in the sporocidal assay. Figure 53 illustrates an exemplary study showing that the exemplary CPO (SEQ
ID NO:2) is effective in decontaminating bacterial spores, using attenuated B. anthracis spores as a model system. The data shows the efficacy of inactivation of attenuated B. anthracis spores by CPO-generated ClO2 (chlorine dioxide). As demonstrated by the data illustrated in Figure 53, the exemplary CPO SEQ ID NO:2 completely killed anthrax spores over a concentration range of eight (8) orders of magnitude.
Example 7: Decontamination of G agents by OPAA enzymes
The invention provides compositions (e.g., enzymes and formulations) and methods for decontaminating, detoxifying and/or neutralizing G agents, such as Soman, Sarin, cyclosarin. In one aspect, the compositions and methods of the invention use an organophosphoric acid anhydrolase (OPAA), such as the exemplary polypeptide of the invention SEQ ID NO: 194, which as OPAA activity, for decontaminating, detoxifying and/or neutralizing G agents. This example demonstrate the decontamination of the G agent Soman (GD) by the exemplary OPAA of the invention SEQ ID NO: 194.
The exemplary OPAA (SEQ ID NO: 194) in a 50 ml reactor decontaminated Soman (GD 1 % w/v): 50 ml side-by-side reactors were used for decontamination of GD at 1% w/v. GD (0.5 g) was dissolved in 50 ml total volume of 0.2 M Bis Tris propane pH 7.2 either with or without purified OPAA (SEQ ID NO: 194 at 100 mg, 2 mg/ml). The reactions were performed in two side-by-side 3-necked 125 ml glass reaction flasks equipped with a mechanical stirring rod (w/ Teflon wing), digital thermometer and polypropylene sampling tube connected to a disposable 1 ml syringe. The reaction flask was kept at constant temperature 200C to 220C by using a thermostatic water reservoir unit connected to plastic tubes circulating the external glass jacket of the reaction flask. The stirring speed was 300 rpm to 340 rpm and samples were taken at specified time intervals. Each sample was immediately diluted in cold DDW to 10"3, 10"5 and 10"6 M. The diluted samples were analyzed by AChE inhibition assay and GC/FPD. Samples were extracted by methyl t-butyl ether (MTBE/water 1:1) before GC analysis. In summary, one reactor contained GD l%w/v dissolved in buffer with OPAA and contained GD solution in buffer only (0.25 M BTP, 0.5 M NaCl, 1 mM MnCl2). Samples were withdrawn from the reaction mixture using a disposable syringe. Temperature was controlled by a thermostatic unit and measured throughout the whole decontamination process (21± I0C).
A time-course of GD (1% w/v) detoxification by OPAA (2 mg/ml) was done using the AChE residual inhibition assay. It was noted that GD was >99.9% detoxified already within the first 8 min (Fig. 2, ti/2 = 1.7 min). It was also noted that the pH remained constant (7-7.5) throughout the enzymatic and non-enzymatic hydrolysis of GD (buffer: Bis Tris propane 0.25M pH=7.2). The level of GD detoxification was measured at enhanced resolution: inhibition of AChE by GD was measured at three different dilutions of GD taken from the 1% w/v reaction mixture that provided the following final concentrations of non-hydrolyzed GD with AChE: 10"8, 10"7 and 10"5 M.
Figure imgf000222_0001
Based on this approach we could demonstrate 99.99% and 99.999% detoxification of GD within 15 and 20 min, respectively (analysis of GD (1% w/v) detoxification by OPAA in a 50 ml reactor by sampling GD solutions at various initial concentrations):
Figure imgf000222_0002
Figure imgf000223_0001
The very high level of GD detoxification (>99.99%) measured by the AChE inhibition assay indicated degradation of the non-toxic P(+)C(+) and P(+)C(-) diastereo- isomers as well. GC analysis demonstrated degradation of all isomers already at 8-15 min. The rate of GD detoxification obtained in a 50 ml reactor was faster and the level of final degradation was higher than those obtained at 0.5 ml reaction volume with GD (1% w/v). The GC analysis of GD during enzymatic degradation corroborated the results obtained from the AChE inhibition assay. The kinetics of GD degradation was analyzed by GC: GD was 99, 99.5 and 99.6% degraded within 5, 8 and 15 min, respectively. The initial decrease in GD concentrations in the control buffer reaction may stem from lower dispersion of GD at the initial period.
From chromatograms of the GC analysis of GD, it could be seen that the peak of GD is composed essentially of four components that represent the four diastereoisomers of GD. Some of the peak shoulders disappear faster than the others during hydrolysis.
Figure 54 illustrates data from a time-course of GD (1% w/v) detoxification by OPAA (2 mg/ml) using the AChE residual inhibition assay, discussed above:
Figure imgf000223_0002
Figure 55 illustrates data from a gas chromatograph analysis of the time-course of GD (1% w/v) detoxification by OPAA (2 mg/ml), discussed above:
Figure imgf000223_0003
Example 8: Decontamination of H agents by dehalogenase enzymes
The invention provides compositions (e.g., enzymes and formulations) and methods for decontaminating, detoxifying and/or neutralizing H agents, such as mustard gas (HD). In one aspect, the compositions and methods of the invention use a dehalogenase (DH), such as the exemplary polypeptide of the invention SEQ ID NO:70, which has dehalogenase activity, for decontaminating, detoxifying and/or neutralizing H agents such as HD. This example demonstrate the decontamination of the H agent mustard has (HD) by the exemplary dehalogenase of the invention SEQ ID NO: 70.
This study demonstrated enzymatic degradation of HD 1% w/v in a 50 ml reactor by the exemplary polypeptide of the invention SEQ ID NO:70. A time-course of HD (1% w/v) degradation by SEQ ID NO:70 (30 mg/ml) in a 50 ml reactor was done. A parallel reaction was performed in the presence of membranes derived from the same expression system of dehalogenase (DHG) without the protein (at 30 mg/ml). The experimental setup is identical to that described above (see Example 7) for GD hydrolysis in a 50 ml reactor. Samples (0.5 ml) were withdrawn from the reaction vessels at specified time- intervals and immediately extracted with equal volume of cold isooctane and kept on ice. Following phase separation by spinning down at 15,000rpm for 1.5 minutes, the isooctane solutions were further diluted into 10"3, 10"4 and 10"5 M nominal non-hydrolyzed HD. The results of GC analysis performed with these samples indicate low extraction yield (approximately 10 to 20%). We assume that this extraction level was similar in both enzymatic and non-enzymatic reactions based on similarity of the cell lysates in both preparations. HD was 98.6 to 99.2% degraded by the exemplary dehalogenase SEQ ID NO: 70 within 15 min compared to approximately 40% in the negative control reaction (see data table, below). These results are similar to those obtained at small reaction volume (0.5 ml). The extraction yield with isooctane using small volume (0.5 ml) in the reaction is significantly higher, since we sample the whole volume of reaction (0.5 ml) and extract it with equal volume of isooctane whereas only 1% of the whole volume is sampled from the 50 ml reactor.
Negative Control DHG SEQ ID NO:70
Figure imgf000224_0001
Figure imgf000225_0001
These values were derived from isooctane solution with 10-fold higher HD concentration (1 mM non hydrolyzed HD)
2 These values were derived from isooctane solution with 5-fold higher HD concentration (0.5 mM non hydrolyzed HD)
3 undetectable (lower than 5xlO'8 M in the original sample)
Another study - a time-course of HD degradation by the exemplary dehalogenase of the invention SEQ ID NO:70, in a 50 ml reactor - was done. This reaction was performed with 0.5 g HD in 50 ml 0.2 M phosphate pH 7.5 containing 10% PEG400 either with negative control or SEQ ID NO:70 (30 mg/ml) at 21± I0C. Samples (0.5 ml) were taken from the reactor and extracted with 0.5 ml isooctane. After phase separation (spun at 15,000 rpm, 1.5 min) isooctane solutions were diluted to 0.1 mM non-hydro lyzed HD before injection on GC column. Samples diluted to 0.1 mM HD could provide detection limit of 0.25% residual HD (99.75% degradation). Thus, 0.5 and 1 mM initial HD concentration provide detection of 99.95 and 99.975% degradation, respectively. Figure 56 illustrates the following data from these studies (where SEQ ID NO: 70 is designated "DHG 5137", and the negative control is "NEG 5123"):
Figure imgf000225_0002
The degradation rate of HD (2% w/v) by the exemplary SEQ ID NO:70 (at 30 mg/ml) was tested in the presence of 10% PEG-400 in 0.2 M phosphate pH 7.5. The total volume of the reaction mixture was 0.5 ml (run in triplicates). Each reaction mixture at a volume of 0.5 ml was extracted entirely at each time interval (0.5, 5, 15, 30 and 60 min) by two consecutive additions of 0.5ml and 0.35 ml isooctane. Quantitative GC/PFPD analysis was performed on HD reaction samples that were further diluted in isooctane (non-hydrolyzed HD concentration of 10"5 M) before injection to GC. Data indicate 75% and 76% degradation of HD by the exemplary SEQ ID NO:70 (30 mg/ml) at 15 and 30 min, respectively. In contrast, HD degradation by negative control was only 24% and 39% at the same time intervals. HD was 97% degraded only after 60 min (compared to 44% with negative control).
Figure imgf000226_0001
Time-course of 2% w/v HD degradation by the exemplary SEQ ID NO:70; reaction volume 0.5 ml using a magnetic stirring, SEQ ID NO:70 at 30 mg/ml, negative control at 30 mg/ml, phosphate 0.2 M pH 7.4, PEG 400 10%v/v, 25°C) triplicates samples of 0.5 ml reaction mixture were extracted twice by 0.5+0.35 ml isooctane and diluted from 0.125 M to 10"5 M HD nominal concentration prior to GC/PFPD analysis.
Example 9: Decontamination of V agents by CPO enzymes
The invention provides compositions (e.g., enzymes and formulations) and methods for decontaminating, detoxifying and/or neutralizing V agents, such as VX gas.
In one aspect, the compositions and methods of the invention use a chloroperoxidase, such as the exemplary polypeptide of the invention SEQ ID NO:2, which has dehalogenase activity, for decontaminating, detoxifying and/or neutralizing H agents such as VX. This example demonstrates the decontamination of the V agent VX by the exemplary chloroperoxidase of the invention SEQ ID NO:2.
A time course of the decontamination of VX was done using 1% VX w/v using the exemplary chloroperoxidase of the invention SEQ ID NO:2 at 1700 u/ml, NaClO2 at
0.4 M, phosphate at 0.3 M, pH at 7.0, at 250C, the data illustrated in Figure 57 (percent
VX detoxification as a function of time) and Figure 58 (pH and time), and the data summarized as:
Figure imgf000227_0001
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. An isolated, synthetic or recombinant nucleic acid comprising (a) a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or complete sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO: 105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO: 119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO: 137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO: 179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO: 189, SEQ ID NO: 191 or SEQ ID NO: 193, over a region of at least about 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues, wherein the nucleic acid encodes at least one polypeptide having an esterase activity, an organophosphoesterase activity, an organophosphohydrolase activity, a carboxylesterase activity, a haloperoxidase activity, a chloroperoxidase (CPO) activity, a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, or encodes a polypeptide or peptide capable of generating an antibody that binds specifically to a polypeptide having a sequence comprising any of the even numbered SEQ ID NO:s in the sequence listing, including from SEQ ID NO:2 through SEQ ID NO: 194, and optionally the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection;
(b) a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO.121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO-.161, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO:187, SEQ ID NO: 189, SEQ ID NO: 191 or SEQ ID NO:193, wherein the nucleic acid encodes at least one polypeptide having an esterase activity, an organophosphoesterase activity, an organophosphohydrolase activity, a carboxylesterase activity, a haloperoxidase activity, a chloroperoxidase (CPO) activity, a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, or encodes a polypeptide or peptide capable of generating an antibody that binds specifically to a polypeptide having a sequence comprising any of the even numbered SEQ ID NO:s in the sequence listing, including from SEQ ID NO:2 through SEQ ID NO: 194; and the stringent conditions include a wash step comprising a wash in 0.2X SSC at a temperature of about 650C for about 15 minutes, and optionally the nucleic acid is at least about 20, 30, 40, 50, 60, 75, 100, 150, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700, 800, 900, 1000 or more residues in length or the full length of the gene or transcript;
(c) a nucleic acid sequence encoding a polypeptide or peptide having a sequence as set forth in 20, 30, 40, 50, 60, 75, 100, 150, 200, 225, 250, 275, 300, 350, 400 or more residues of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID
NO: 102, SEQ ID NO:104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192 or SEQ ID NO:194, wherein the nucleic acid encodes at least one polypeptide having an esterase activity, an organophosphoesterase activity, an organophosphohydrolase activity, a carboxylesterase activity, a haloperoxidase activity, a chloroperoxidase (CPO) activity, a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, or encodes a polypeptide or peptide capable of generating an antibody that binds specifically to a polypeptide having a sequence comprising any of the even numbered SEQ ID NO:s in the sequence listing, including from SEQ ID NO:2 through SEQ ID NO: 194; or
(d) a nucleic acid sequence complementary to (a), (b) or (c).
2. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the nucleic acid sequence comprises a sequence as set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO: 103, SEQ ID
NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO: 113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO: 129, SEQ ID NO:131, SEQ ID NO: 133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO: 139 SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO: 149, SEQ ID NO:151, SEQ ID NO: 153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191 or SEQ ID NO: 193.
3. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the sequence comparison algorithm comprises a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d "nr pataa" -F F, and all other options are set to default.
4. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the enzymatic activity comprises a detoxifying or decontaminating activity.
5. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a V agent.
6. The isolated, synthetic or recombinant nucleic acid of claim 5, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a V agent, or the enzymatic activity comprises a haloperoxidase activity, or an activity comprising catalyzing the hydrolysis of a methylphosphonofluoridate or a thiophosphoric ester, or a combination thereof.
7. The isolated, synthetic or recombinant nucleic acid of claim 6, wherein the haloperoxidase activity comprises a chloroperoxidase activity.
8. The isolated, synthetic or recombinant nucleic acid of claim 5, wherein the V agent comprises VX (0-Ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothioate, or methylphosphonothioic acid), VE (0-Ethyl-S-[2-(diethylamino)ethyl] ethylphosphonothioate), VG (O,O-Diethyl-S-[2-(diethylamino)ethyl] phosphorothioate), VM (O-Ethyl-S-[2-(diethylamino)ethyl] methylphosphonothioate), VR (Phosphonothioic acid) Soviet V-gas (Russian VX), Tetriso (0,0-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate) or a phosphorylthiocholine-comprising compound.
9. The isolated, synthetic or recombinant nucleic acid of claim 5, wherein the enzyme hydrolyzing or decontaminating a V agent comprises a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 , SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO: 89; SEQ ID NO: 117; SEQ ID NO: 1 19; SEQ ID NO: 127; SEQ ID NO: 151 ; SEQ ID NO: 167; SEQ ID NO: 171 ; SEQ ID NO: 187 or SEQ ID NO: 191, or having an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:76; SEQ ID NO:78; SEQ ID NO:90; SEQ ID NO: 1 18; SEQ ID NO: 120; SEQ ID NO: 128; SEQ ID NO: 152; SEQ ID NO: 168; SEQ ID NO: 172; SEQ ID NO: 189 or SEQ ID NO: 192.
10. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a G agent.
11. The isolated, synthetic or recombinant nucleic acid of claim 10, wherein the enzymatic activity comprising hydrolysis of, or decontamination of, a G agent comprises an organophosphoric acid anhydrolase (OPAA) activity.
12. The isolated, synthetic or recombinant nucleic acid of claim 10, wherein the G agent comprises tabun (GA), sarin (methylphosphonofluoridic acid) (GB), soman (GD), cyclosarin (GF) or a combination thereof.
13. The isolated, synthetic or recombinant nucleic acid of claim 5, wherein the enzyme hydrolyzing or decontaminating a G agent comprises a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO:73; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO: 101; SEQ ID NO: 103; SEQ ID
NO:105; SEQ IDNO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ IDNO:113; SEQ ID NO:115; SEQ ID NO: 117; SEQ ID NO:119; SEQ ID NO:121; SEQ ID NO:123; SEQ ID NO:125; SEQ IDNO:127; SEQ IDNO:129; SEQ ID NO:131; SEQ IDNO:133; SEQ ID NO:135; SEQIDNO:137; SEQIDNO:139; SEQIDNO:141; SEQIDNO:143; SEQID NO: 145; SEQIDNO:147; SEQIDNO:149; SEQIDNO:151; SEQIDNO:153; SEQID NO:155; SEQIDNO:157; SEQIDNO:159; SEQIDNO:161; SEQIDNO:163; SEQID NO:165; SEQIDNO:167; SEQIDNO:169; SEQIDNO:171; SEQIDNO:173; SEQID NO:175; SEQIDNO:177; SEQIDNO:179; SEQ IDNO:181; SEQIDNO:183; SEQID NO:185; SEQ ID NO:187 or SEQ ID NO:191; or, a polypeptide having a prolidase activity or an organophosphoric acid anhydrolase (OPAA) activity and encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having a sequence as set forth in SEQ ID NO: 194.
14. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, an H agent.
15. The isolated, synthetic or recombinant nucleic acid of claim 14, wherein the H agent comprises a chloroperoxidase activity, a dehalogenase activity or a combination thereof.
16. The isolated, synthetic or recombinant nucleic acid of claim 14, wherein the H agent comprises mustard gas; 1 1' thiobis [2 chloroethane] bis-(2-chloroethyl) sulphide; beta, beta' dichloroethyl sulphide; 2, 2' dichloroethyl sulphide; bis (beta-chloroethyl) sulphide; l-chloro-2 (beta-chlorodiethylthio) ethane; or, a combination thereof.
17. The isolated, synthetic or recombinant nucleic acid of claim 14, wherein the enzyme hydrolyzing or decontaminating an H agent comprises a polypeptide encoded by a nucleic acid encoding a chloroperoxidase and having a sequence as set forth in SEQ ID NO:1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61;
SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58;
SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively, or a nucleic acid encoding a dehalogenase and having a sequence as set forth in
SEQ ID NO:69 or SEQ ID NO:91 or the enzyme has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92..
18. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a biological agent.
19. The isolated, synthetic or recombinant nucleic acid of claim 18, wherein the enzymatic activity comprises a chloroperoxidase activity.
20. The isolated, synthetic or recombinant nucleic acid of claim 18, wherein the biological agent is a gram negative spore.
21. The isolated, synthetic or recombinant nucleic acid of claim 20, wherein the gram negative spore comprises an anthrax, or Bacillus anthracis, spore.
22. The isolated, synthetic or recombinant nucleic acid of claim 18 wherein the enzyme hydrolyzing or decontaminating a biological agent comprises a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO:1, SEQ ID NO: 53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
23. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a P-F bond.
24. The isolated, synthetic or recombinant nucleic acid of claim 23, wherein the polypeptide having P-F bond hydrolyzing or decontamination activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:71 ; SEQ ID NO:73; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO: 101; SEQ ID NO: 103; SEQ ID NO: 105; SEQ IDNO:107; SEQ ID NO: 109; SEQ ID NO: 111; SEQ ID NO:113;SEQIDNO:115;SEQIDNO:117;SEQIDNO:119;SEQIDNO:121;SEQID NO:123; SEQIDNO:125; SEQIDNO:127; SEQIDNO:129; SEQ IDNO:131; SEQID NO:133; SEQIDNO:135; SEQIDNO:137; SEQIDNO:139; SEQIDNO:141; SEQID NO:143; SEQ IDNO:145; SEQ IDNO:147; SEQ IDNO:149; SEQ IDNO:151; SEQ ID NO:153; SEQIDNO:155; SEQIDNO:157; SEQ IDNO:159; SEQ IDNO:161; SEQID NO:163; SEQIDNO:165; SEQIDNO:167; SEQ ID NO: 169; SEQ ID NO: 171; SEQID NO:173; SEQIDNO:175; SEQIDNO:177; SEQIDNO:179; SEQIDNO:181; SEQID NO:183; SEQIDNO:185; SEQIDNO:187, SEQIDNO:191 or SEQ ID NO: 193, or has an amino acid sequence as set forth in SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO: 100; SEQ ID NO: 102; SEQ ID NO: 104; SEQ ID NO: 106; SEQ ID NO: 108; SEQ ID NO: 110; SEQ ID NO: 112; SEQ ID NO: 114; SEQIDNO:116; SEQIDNO:118; SEQIDNO:120; SEQIDNO:122; SEQIDNO:124; SEQ ID NO: 126; SEQ ID NO: 128; SEQ ID NO: 130; SEQ ID NO: 132; SEQ ID NO: 134; SEQ ID NO: 136; SEQ ID NO: 138; SEQ ID NO: 140; SEQ ID NO: 142; SEQ ID NO: 144; SEQ ID NO: 146; SEQ ID NO: 148; SEQ ID NO: 150; SEQ ID NO: 152; SEQ ID NO: 154; SEQ ID NO: 156; SEQ ID NO: 158; SEQ ID NO: 160; SEQ ID NO: 162; SEQ ID NO: 164; SEQ ID NO: 166; SEQ ID NO: 168; SEQ ID NO: 170; SEQ ID NO: 172; SEQ ID NO: 174; SEQ ID NO: 176; SEQ ID NO: 178; SEQ ID NO: 180; SEQ ID NO: 182; SEQ ID NO: 184; SEQ ID NO:186; SEQ ID NO:188, SEQ ID NO:192 or SEQ IDNO:194.
25. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a P-S bond.
26. The isolated, synthetic or recombinant nucleic acid of claim 25, wherein the polypeptide having P-S bond hydrolyzing or decontamination activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:1; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 89; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO:127; SEQ ID
NO: 151; SEQ ID NO:167; SEQ ID NO:171; SEQ ID NO:187 or SEQ ID NO:191, or has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:76; SEQ ID NO: 78; SEQ ID NO:90; SEQ ID NO: 118; SEQ ID NO: 120; SEQ ID NO: 128; SEQ ID NO: 152; SEQ ID NO: 168; SEQ ID NO: 172; SEQ ID NO: 188 or SEQ ID NO: 192.
27. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the enzymatic activity comprises an organophosphoric acid anhydrolase (OPAA) activity.
28. The isolated, synthetic or recombinant nucleic acid of claim 27, wherein the enzyme having organophosphoric acid anhydrolase (OPAA) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or has an amino acid sequence as set forth in SEQ ID NO: 194.
29. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises an aminopeptidase activity.
30. The isolated, synthetic or recombinant nucleic acid of claim 27, wherein the enzyme having aminopeptidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 189, or has an amino acid sequence as set forth in SEQ ID NO: 190.
31. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the enzymatic activity comprises a carboxylesterase activity.
32. The isolated, synthetic or recombinant nucleic acid of claim 27, wherein the enzyme having carboxylesterase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:73; SEQ ID NO.75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81 ; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87 or SEQ ID NO:89.
33. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises a heme-based peroxidase activity.
34. The isolated, synthetic or recombinant nucleic acid of claim 33, wherein the enzyme having heme-based peroxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID
NO:51, or, the enzyme has a sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO.8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively.
35. The isolated, synthetic or recombinant nucleic acid of claim 34, wherein the enzyme has a heme-based chloroperoxidase activity and is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1, or has an amino acid sequence as set forth in SEQ ID NO:2.
36. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises a non-heme-based peroxidase activity.
37. The isolated, synthetic or recombinant nucleic acid of claim 37, wherein the enzyme having non-heme-based chloroperoxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
38. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises a dehalogenase activity.
39. The isolated, synthetic or recombinant nucleic acid of claim 38, wherein the enzyme having dehalogenase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
40. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises a diisopropylfluorophosphatase (DFPase) activity.
41. The isolated, synthetic or recombinant nucleic acid of claim 38, wherein the enzyme having diisopropylfluorophosphatase (DFPase) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72.
42. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises an organophosphoesterase and can hydrolyze a P-S or a P-F bond and detoxify an acetylcholinesterase- or butyrylcholinesterase- inhibitor.
43. The isolated, synthetic or recombinant nucleic acid of claim 1 , wherein the enzymatic activity comprises a halogenation reaction to form a hypohalite from hydrogen peroxide and chloride, bromide or iodide.
44. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises catalysis of the transfer of oxygen from hydrogen peroxide to an organic substrate.
45. The isolated, synthetic or recombinant nucleic acid of claim 44, wherein the enzymatic activity comprises chemical bleaching of lignin.
46. The isolated, synthetic or recombinant nucleic acid of claim 45, wherein the enzymatic activity comprises chemical bleaching of lignin in a pulp or paper manufacturing process.
Figure imgf000239_0001
n υ
47. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the enzymatic activity comprises detoxifying a pesticide, herbicide and/or insecticide.
48. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the pesticide is Demeton-S, Demeton-S-methyl, Demeton-S-methylsulphon, Demeton- methyl, Parathion, Phosmet, Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos- ethyl, Amiton, Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton, Endothion, Etion, Ethoate-methyl, Formothion, Malathion, Mercarbam, Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone, Prothidathion, Prothoate, Sophamide, Thiometon, Vamidothion, Methamidophos or a combination thereof.
49. The isolated, synthetic or recombinant nucleic acid of any one of claims 1 to 48, wherein the nucleic acid encodes a thermostable or thermotolerant enzyme or protein.
50. The isolated, synthetic or recombinant nucleic acid of claim 49, wherein the enzyme or protein retains activity under conditions comprising a temperature range of between about 37°C to about 95°C, or between about 55°C to about 85°C, or between about 700C to about 750C, or between about 7O0C to about 95°C, or between about 9O0C to about 950C, or, retains enzyme activity under conditions comprising a temperature range of from greater than 370C to about 95°C, from greater than 550C to about 85°C, or between about 7O0C to about 750C, or from greater than 900C to about 95°C, or in the range between about I0C to about 5°C, between about 50C to about 15°C, between about 150C to about 25°C, between about 25°C to about 37°C, between about 370C to about 950C.
51. The isolated, synthetic or recombinant nucleic acid of claim 49, wherein the enzyme or protein retains an activity after exposure to a temperature in the range from greater than 370C to about 950C, from greater than 55°C to about 85°C, from greater than 900C to about 950C, or after exposure to a temperature in the range between about 1°C to about 5°C, between about 5°C to about 150C, between about 150C to about 250C, between about 250C to about 37°C, between about 37°C to about 95°C, between about 55°C to about 85°C, between about 700C to about 750C, or between about 900C to about 95°C.
52. A nucleic acid probe for identifying a nucleic acid encoding a polypeptide with a hydrolase activity, an esterase activity, an organophosphohydrolase activity, an organophosphoesterase activity, a carboxylesterase activity, a haloperoxidase activity, a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity or an organophosphoric acid anhydrolase (OPAA) activity, wherein the probe comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more consecutive bases of: (a) a sequence as set forth in claim 1, or
(b) a nucleic acid comprising at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more consecutive residues of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO.27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO.47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO: 123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO: 131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO: 153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO.171, SEQ ID NO-.173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO.189, SEQ ID NO: 191 or SEQ ID NO: 193; wherein the probe identifies the nucleic acid by binding or hybridization.
53. The nucleic acid probe of claim 52, wherein the probe comprises an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, about 60 to 100, or about 50 to 150 consecutive bases.
54. An amplification primer pair for amplifying a nucleic acid encoding a polypeptide having an enzymatic activity or encoding a protein, wherein the primer pair is capable of amplifying a nucleic acid comprising
(a) a sequence as set forth in claim 1, or a subsequence thereof; or
(b) a nucleic acid comprising at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more consecutive residues of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO.83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID
NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO:109, SEQ ID NO: 111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO.151, SEQ ID NO: 153, SEQ ID NO.155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO: 163, SEQ ID NO.165, SEQ ID NO:167, SEQ ID NO.169, SEQ ID NO:171, SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 177, SEQ ID NO:179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO: 191 or SEQ ID NO: 193, wherein a member of the amplification primer pair comprises an oligonucleotide comprising at least about 10 to 50 consecutive bases of the sequence, or, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40 or more consecutive bases of the sequence.
55. The amplification primer pair of claim 54, wherein the amplification primer pair amplifies a nucleic acid encoding a polypeptide with a hydrolase activity, an esterase activity, an organophosphohydrolase activity, an organophosphoesterase activity, a carboxylesterase activity, a haloperoxidase activity, a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity or an organophosphoric acid anhydrolase (OPAA) activity.
56. The amplification primer pair of claim 54, comprising a first member having a sequence as set forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 42, 33, 34, 35 or more residues of the nucleic acid of (a) or (b), and a second member having a sequence as set forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 42, 33, 34, 35 or more residues of the complementary strand of the first member.
57. An enzyme-encoding or protein-encoding nucleic acid generated by amplification of a polynucleotide using an amplification primer pair as set forth in any of claims 54 to 56.
58. The enzyme-encoding or protein-encoding nucleic acid of claim 57, wherein the amplification is by a polymerase chain reaction (PCR).
59. The enzyme-encoding or protein-encoding nucleic acid of claim 57, wherein the nucleic acid is generated by amplification of a gene library, and optionally the gene library is an environmental library.
60. An isolated, synthetic or recombinant polypeptide having an enzymatic activity or encoding a protein encoded by a nucleic acid having a sequence as set forth in claim 57.
61. A method of amplifying a nucleic acid encoding a polypeptide having an enzymatic activity or encoding a protein comprising amplification of a template nucleic acid with an amplification primer pair as set forth in any of claims 54 to 56, wherein the amplification primer pair is capable of amplifying a nucleic acid sequence as set forth in claim 1 , or a subsequence thereof.
62. An expression cassette comprising a nucleic acid comprising a sequence as set forth in claim 1 or claim 57.
63. A vector comprising a nucleic acid comprising a sequence as set forth in claim 1 or claim 57.
64. A cloning vehicle comprising a nucleic acid comprising a sequence as set forth in claim 1 or claim 57, wherein the cloning vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome.
65. The cloning vehicle of claim 64, wherein the viral vector comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector.
66. A bacterial artificial chromosome (BAC), a bacteriophage Pl -derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC) comprising a sequence as set forth in claim 1 or claim 57.
67. A transformed cell comprising a nucleic acid comprising: a sequence as set forth in claim 1 or claim 57; or, an expression cassette as set forth in claim 62, a cloning vehicle as set forth in claim 64, or a bacterial artificial chromosome (BAC), a bacteriophage Pl -derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC) as set forth in claim 66.
68. The transformed cell of claim 67, wherein the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
69. A transgenic non-human animal comprising a sequence as set forth in claim 1 or claim 57, and optionally the animal is a mouse, a goat, a rabbit, a sheep, a pig, a cow or a rat.
70. A transgenic plant comprising a sequence as set forth in claim 1 or claim 57, and optionally the plant is a corn plant, a sorghum plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant, a grass, or a tobacco plant.
71. A transgenic seed comprising a sequence as set forth in claim 1 or claim 57, and optionally the seed is a corn seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a rice, a barley, a peanut or a tobacco plant seed.
72. An antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a sequence as set forth in claim 1 or claim 57, or a subsequence thereof, and optionally the antisense oligonucleotide has a length of between about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 bases in length.
73. A method of inhibiting the translation of an enzyme-encoding or protein- encoding message in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a sequence as set forth in claim 1 or claim 57.
74. A double-stranded inhibitory RNA (RNAi) molecule comprising a subsequence of a sequence as set forth in claim 1 or claim 57, wherein optionally the RNAi is an siRNA or an miRNA molecule, or optionally the RNAi is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length, wherein optionally the RNAi is an siRNA or an miRNA molecule.
75. A method of inhibiting the expression of an enzyme or a protein in a cell comprising administering to the cell or expressing in the cell a double-stranded inhibitory RNA (iRNA) as set forth in claim 74.
76. An isolated, synthetic or recombinant polypeptide or peptide: (a) having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or complete sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO: 72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO:104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO: 126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO: 138, SEQ ID NO:140, SEQ ID NO: 142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO.150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO.192 or SEQ ID NO: 194, over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues, wherein optionally the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection, or, (ii) encoded by a nucleic acid as set forth in claim 1 ; wherein the polypeptide or peptide of (i) or (ii) has an esterase activity, an organophosphoesterase activity, an organophosphohydrolase activity, a carboxylesterase activity, a haloperoxidase activity, a chloroperoxidase (CPO) activity, a heme-based (hCPO) or a non-heme chloroperoxidase (nhCPO) activity, a diisopropylfluorophosphatase (DFPase) activity, a dehalogenase activity, an oxidoreductase activity, a prolidase activity, an imidodipeptidase activity and/or an organophosphoric acid anhydrolase (OPAA) activity, or encodes a polypeptide or peptide capable of generating an antibody that binds specifically to a polypeptide having a sequence comprising any of the even numbered SEQ ID NO:s in the sequence listing, including from SEQ ID NO:2 through SEQ ID NO: 194.
77. The isolated, synthetic or recombinant nucleic acid of claim 1, wherein the sequence comparison algorithm comprises a BLAST version 2.2.2 algorithm where a filtering setting is set to blastall -p blastp -d "nr pataa" -F F, and all other options are set to default.
78. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises a detoxifying or decontaminating activity.
79. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a V agent.
80. The isolated, synthetic or recombinant polypeptide or peptide of claim 79, wherein the enzymatic activity comprising hydrolysis of, or decontamination of, a V agent comprises a haloperoxidase activity, an activity comprising catalyzing the hydrolysis of a methylphosphonofluoridate or a thiophosphoric ester, or a combination thereof.
81. The isolated, synthetic or recombinant polypeptide or peptide of claim 80, wherein the haloperoxidase activity comprises a chloroperoxidase activity.
82. The isolated, synthetic or recombinant polypeptide or peptide of claim 79, wherein the V agent comprises VX (0-Ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothioate, or methylphosphonothioic acid), VE (O-Ethyl-S-[2- (diethylamino)ethyl] ethylphosphonothioate), VG (O,O-Diethyl-S-[2- (diethylamino)ethyl] phosphorothioate), VM (O-Ethyl-S-[2-(diethylamino)ethyl] methylphosphonothioate), VR (Phosphonothioic acid) Soviet V-gas (Russian VX), Tetriso (0,0-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate) or a phosphorylthiocholine-comprising compound.
83. The isolated, synthetic or recombinant polypeptide or peptide of claim 79, wherein the enzyme hydrolyzing or decontaminating a V agent comprises a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 89; SEQ ID NO: 117; SEQ IDNO:119; SEQ ID NO.127; SEQ ID NO:151; SEQ ID NO:167; SEQ ID NO:171; SEQ IDNO:187 or SEQ IDNO:191.
84. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a G agent.
85. The isolated, synthetic or recombinant polypeptide or peptide of claim 84, wherein the enzymatic activity comprising hydrolysis of, or decontamination of, a G agent comprises an organophosphoric acid anhydrolase (OPAA) activity, or, diisopropylfluorophosphatase (DFPase) activity.
86. The isolated, synthetic or recombinant polypeptide or peptide of claim 84, wherein the G agent comprises tabun (GA), sarin (methylphosphonofluoridic acid) (GB), soman (GD), cyclosarin (GF) or a combination thereof.
87. The isolated, synthetic or recombinant polypeptide or peptide of claim 84, wherein the enzyme hydrolyzing or decontaminating a G agent comprises a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO: 101; SEQ ID NO: 103; SEQ ID NO: 105; SEQ ID NO: 107; SEQ ID NO: 109; SEQ ID NO:111; SEQ ID NO:113; SEQ ID NO: 115; SEQ ID NO: 117; SEQ ID NO:119; SEQ ID NO: 121; SEQ IDNO:123; SEQIDNO:125; SEQIDNO:127; SEQIDNO:129; SEQIDNO:131; SEQ IDNO:133; SEQIDNO:135; SEQIDNO:137; SEQIDNO:139; SEQIDNO:141; SEQ ID NO: 143; SEQ ID NO: 145; SEQ ID NO: 147; SEQ ID NO: 149; SEQ ID NO: 151 ; SEQ IDNO:153; SEQIDNO:155; SEQIDNO:157; SEQIDNO:159; SEQIDNO:161; SEQ IDNO:163; SEQIDNO:165; SEQIDNO:167; SEQ ID NO: 169; SEQ ID NO: 171; SEQ IDNO:173; SEQIDNO:175; SEQIDNO:177; SEQIDNO:179; SEQIDNO:181; SEQ IDNO:183; SEQIDNO:185; SEQIDNO:187, or SEQ IDNO:191; or has an amino acid sequence as set forth in SEQ ID NO:72; SEQ ID NO:74; SEQ ID NO:94; SEQ ID NO:96; SEQ ID NO:98; SEQ ID NO: 100; SEQ ID NO: 102; SEQ ID NO: 104; SEQ ID NO: 106; SEQIDNO:108; SEQIDNO:110; SEQIDNO:112; SEQIDNO:114; SEQIDNO:116; jOHtυ-zu itu.tυ/ uzz /υ-i w υ
SEQ ID NO:118; SEQ ID NO:120; SEQ ID NO: 122; SEQ ID NO:124; SEQ ID NO:126;
SEQ ID NO:128; SEQ ID NO:130; SEQ ID NO: 132; SEQ ID NO:134; SEQ ID NO.136;
SEQ ID NO: 138; SEQ ID NO: 140; SEQ ID NO: 142; SEQ ID NO: 144; SEQ ID NO: 146;
SEQ ID NO: 148; SEQ ID NO: 150; SEQ ID NO: 152; SEQ ID NO: 154; SEQ ID NO: 156; SEQ ID NO:158; SEQ ID NO.160; SEQ ID NO: 162; SEQ ID NO:164; SEQ ID NO.166;
SEQ ID NO: 168; SEQ ID NO: 170; SEQ ID NO: 172; SEQ ID NO: 174; SEQ ID NO: 176;
SEQ ID NO: 178; SEQ ID NO: 180; SEQ ID NO: 182; SEQ ID NO: 184; SEQ ID NO: 186;
SEQ ID NO: 188, or SEQ ID NO: 192, or, a polypeptide having a prolidase activity or an organophosphoric acid anhydrolase (OPAA) activity and encoded by a nucleic having a sequence as set forth in
SEQ ID NO: 193, or has an amino acid sequence as set forth in SEQ ID NO: 194; or, a polypeptide having diisopropyl-fluorophosphatase (DFPase) activity encoded by a nucleic having a sequence as set forth in SEQ ID NO:71 , or has an amino acid sequence as set forth in SEQ ID NO:72.
88. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, an H agent.
89. The isolated, synthetic or recombinant polypeptide or peptide of claim 88, wherein the H agent comprises a chloroperoxidase activity, a dehalogenase activity or a combination thereof.
90. The isolated, synthetic or recombinant polypeptide or peptide of claim 88, wherein the H agent comprises mustard gas; 1 1' thiobis [2 chloroethane] bis-(2- chloroethyl) sulphide; beta, beta' dichloroethyl sulphide; 2, 2' dichloroethyl sulphide; bis (beta-chloroethyl) sulphide; 1 -chloro-2 (beta-chlorodiethylthio) ethane; or, a combination thereof.
91. The isolated, synthetic or recombinant polypeptide or peptide of claim 88, wherein the enzyme hydrolyzing or decontaminating an H agent comprises a polypeptide encoded by a nucleic acid encoding a chloroperoxidase and having a sequence as set forth in SEQ ID NO: 1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61 ; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO.66 or SEQ ID NO:68, respectively, or a nucleic acid encoding a dehalogenase and having a sequence as set forth in SEQ ID NO:69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
92. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a biological agent.
93. The isolated, synthetic or recombinant polypeptide or peptide of claim 92, wherein the enzymatic activity comprises a haloperoxidase activity.
94. The isolated, synthetic or recombinant polypeptide or peptide of claim 93, wherein the haloperoxidase activity comprises a chloroperoxidase activity.
95. The isolated, synthetic or recombinant polypeptide or peptide of claim 92, wherein the biological agent is a gram negative spore.
96. The isolated, synthetic or recombinant polypeptide or peptide of claim 94, wherein the gram negative spore comprises an anthrax, or Bacillus anthracis, spore.
97. The isolated, synthetic or recombinant polypeptide or peptide of claim 92, wherein the enzyme hydrolyzing or decontaminating a biological agent comprises a polypeptide encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 ; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
98. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a P-F bond.
99. The isolated, synthetic or recombinant polypeptide or peptide of claim 98, wherein the polypeptide having P-F bond hydrolyzing or decontamination activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:73; SEQ ID NO:93; SEQ ID NO:95; SEQ ID NO:97; SEQ ID NO:99; SEQ ID NO:101; SEQ ID NO:103; SEQ ID NO:105; SEQ ID NO:107; SEQ ID NO:109; SEQ ID NO:111; SEQ ID NO.113; SEQ ID NO:115; SEQ ID NO:117; SEQ ID NO:119; SEQ IDNO:121; SEQ ID NO:123; SEQ ID NO: 125; SEQ ID NO: 127; SEQ ID NO: 129; SEQ ID NO: 131; SEQ ID NO: 133; SEQ ID NO:135; SEQ ID NO:137; SEQ ID NO:139; SEQ ID NO:141; SEQ ID NO:143; SEQ ID NO:145; SEQ ID NO:147; SEQ ID NO:149; SEQ ID NO:151; SEQ ID NO:153; SEQ ID NO:155; SEQ ID NO:157; SEQ ID NO:159; SEQ IDNO:161; SEQ ID NO.163; SEQ ID NO:165; SEQ ID NO:167; SEQ ID NO:169; SEQ IDNO:171; SEQ IDNO:173; SEQ ID NO:175; SEQ ID NO:177; SEQ ID NO:179; SEQ IDNO:181; SEQ IDNO.183; SEQ ID NO: 185; SEQ ID NO: 187 or SEQ ID NO: 191.
100. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises hydrolysis of, or decontamination of, a P-S bond.
101. The isolated, synthetic or recombinant polypeptide or peptide of claim 100, wherein the polypeptide having P-S bond hydrolyzing or decontamination activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 ; SEQ ID NO: 75;
SEQ ID NO: 77; SEQ ID NO: 89; SEQ ID NO:117; SEQ ID NO:119; SEQ ID NO: 127;
SEQ ID NO:151; SEQ ID NO:167; SEQ ID NO: 171; SEQ ID NO:187 or SEQ ID
NO:191.
102. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises an organophosphoric acid anhydrolase (OPAA) activity.
103. The isolated, synthetic or recombinant polypeptide or peptide of claim
102, wherein the enzyme having organophosphoric acid anhydrolase (OPAA) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194. OOttu-z.υ i tυ.tu/
Figure imgf000251_0001
i vy \j
104. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises an aminopeptidase activity.
105. The isolated, synthetic or recombinant polypeptide or peptide of claim 104, wherein the enzyme having aminopeptidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 189, or having an amino acid sequence as set forth in SEQ ID NO: 190.
106. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises a carboxylesterase activity.
107. The isolated, synthetic or recombinant polypeptide or peptide of claim 106, wherein the enzyme having carboxylesterase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87 or SEQ ID NO:89.
108. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises a heme-based peroxidase activity.
109. The isolated, synthetic or recombinant polypeptide or peptide of claim 108, wherein the enzyme having heme-based peroxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO.l l; SEQ ID NO:13; SEQ ID NO: 15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31 ; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO:12; SEQ ID NO: 14; SEQ ID NO:16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively.
110. The isolated, synthetic or recombinant polypeptide or peptide of claim 109, wherein the enzyme has a heme-based chloroperoxidase activity and is encoded by a nucleic having a sequence as set forth in SEQ ID NO:1, or has an amino acid sequence as set forth in SEQ ID NO:2.
111. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises a non-heme-based peroxidase activity.
112. The isolated, synthetic or recombinant polypeptide or peptide of claim 111, wherein the enzyme having non-heme-based chloroperoxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO: 65 or SEQ ID NO: 67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
113. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises a dehalogenase activity.
114. The isolated, synthetic or recombinant polypeptide or peptide of claim
1 13, wherein the enzyme having dehalogenase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 69 or SEQ ID NO:91, or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO:92, respectively.
115. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises a diisopropylfluorophosphatase (DFPase) activity.
116. The isolated, synthetic or recombinant polypeptide or peptide of claim 1 15, wherein the enzyme having diisopropylfluorophosphatase (DFPase) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO: 72. jυi-Tvj-z-u i -ru .Tυ/ ivz.^. ι \j~ ι vv yj
117. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises an organophosphoesterase and can hydrolyze a P-S or a P-F bond and detoxify an acetylcholinesterase- or butyrylcholinesterase- inhibitor.
118. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises a halogenation reaction to form a hypohalite from hydrogen peroxide and chloride, bromide or iodide.
119. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises catalysis of the transfer of oxygen from hydrogen peroxide to an organic substrate.
120. The isolated, synthetic or recombinant polypeptide or peptide of claim 119, wherein the enzymatic activity comprises chemical bleaching of lignin.
121. The isolated, synthetic or recombinant polypeptide or peptide of claim 120, wherein the enzymatic activity comprises chemical bleaching of lignin in a pulp or paper manufacturing process.
122. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the enzymatic activity comprises detoxifying a pesticide, herbicide and/or insecticide.
123. The isolated, synthetic or recombinant polypeptide or peptide of claim
122, wherein the pesticide is Demeton-S, Demeton-S-methyl, Demeton-S-methylsulphon, Demeton-methyl, Parathion, Phosmet, Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos-ethyl, Amiton, Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton, Endothion, Etion, Ethoate-methyl, Formothion, Malathion, Mercarbam, Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone, Prothidathion, Prothoate, Sophamide, Thiometon, Vamidothion, Methamidophos or a combination thereof. JUtTVJ-Z-U l tu .tvj/ iy/.z. / u- 1 vv vy
124. The isolated, synthetic or recombinant polypeptide or peptide of any one of claims 76 to 123, wherein the nucleic acid encodes a thermostable or thermotolerant enzyme or protein.
125. The isolated, synthetic or recombinant polypeptide or peptide of claim
124, wherein the polypeptide retains enzyme activity under conditions comprising a temperature range of between about 37°C to about 95°C, or between about 55°C to about 85°C, or between about 700C to about 75°C, or between about 700C to about 95°C, or between about 900C to about 95°C, or, retains enzyme activity under conditions comprising a temperature range of from greater than 37°C to about 950C, from greater than 55°C to about 85°C, or between about 700C to about 750C, or from greater than 900C to about 95°C, or in the range between about 1°C to about 50C, between about 5°C to about 15°C, between about 15°C to about 25°C, between about 25°C to about 370C, between about 37°C to about 95°C.
126. The isolated, synthetic or recombinant polypeptide or peptide of claim 124, wherein the polypeptide retains an activity after exposure to a temperature in the range from greater than 37°C to about 950C, from greater than 55°C to about 85°C, from greater than 900C to about 950C, or after exposure to a temperature in the range between about rC to about 50C, between about 5°C to about 15°C, between about 15°C to about 250C, between about 25°C to about 370C, between about 37°C to about 95°C, between about 550C to about 85°C, between about 700C to about 75°C, or between about 900C to about 95°C.
127. An isolated, synthetic or recombinant polypeptide or peptide comprising a sequence as set forth in claim 76 and lacking a signal sequence.
128. An isolated, synthetic or recombinant polypeptide or peptide comprising a sequence as set forth in claim 76 and having a heterologous signal sequence.
129. The isolated, synthetic or recombinant polypeptide or peptide of claim 76 having a specific activity at about 370C in the range from about 100 to about 1000 units per milligram of protein, from about 500 to about 750 units per milligram of protein, from jottu--,υ itu.tυ/ Lf-.4 / υ- i vv vj
about 500 to about 1200 units per milligram of protein, or from about 750 to about 1000 units per milligram of protein.
130. The isolated, synthetic or recombinant polypeptide or peptide of claim 124, wherein the thermotolerance comprises retention of at least half of the specific activity of the hydrolase at 37°C after being heated to an elevated temperature.
131. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the polypeptide comprises at least one glycosylation site, and optionally the glycosylation is an N-linked glycosylation, and optionally the polypeptide is glycosylated after being expressed in a P. pastoris or a S. potnbe.
132. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the polypeptide or peptide retains enzyme or binding activity under conditions comprising about pH 6.5, pH 6.0, pH 5.5, 5.0, pH 4.5 or 4.0.
133. The isolated, synthetic or recombinant polypeptide or peptide of claim 76, wherein the polypeptide or peptide retains enzyme or binding activity under conditions comprising about pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10 or pH 10.5.
134. A protein preparation comprising a polypeptide or peptide of claim 76, wherein the protein preparation comprises a liquid, a solid or a gel.
135. A heterodimer comprising a polypeptide or peptide of claim 76 and a second domain, and optionally the second domain is an epitope or a tag.
136. The heterodimer of claim 135, wherein the second domain is a polypeptide and the heterodimer is a fusion protein.
137. A homodimer comprising a polypeptide or peptide of claim 76.
138. An immobilized polypeptide or peptide, wherein the polypeptide or peptide comprises a sequence as set forth in claim 76, or a subsequence thereof. juttυ-iυ itu.tu/ uz.4. / u- i vy \j
139. The immobilized polypeptide or peptide of claim 138, wherein the polypeptide or peptide is immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.
140. An array comprising an immobilized polypeptide or peptide as set forth in claim 138 or claim 139.
141. An array comprising an immobilized nucleic acid as set forth in claim 1 or claim 57.
142. An isolated, synthetic or recombinant antibody that specifically binds to a polypeptide or peptide as set forth in claim 76, and optionally the antibody is a monoclonal or a polyclonal antibody.
143. A hybridoma comprising an antibody that specifically binds to a polypeptide as set forth in claim 76.
144. A method of isolating or identifying a polypeptide with an enzymatic activity or encoding a protein comprising the steps of:
(a) providing an antibody as set forth in claim 142;
(b) providing a sample comprising polypeptides; and
(c) contacting the sample of step (b) with the antibody of step (a) under conditions wherein the antibody can specifically bind to the polypeptide, thereby isolating or identifying a polypeptide having enzymatic activity or encoding a protein.
145. A method of making an anti-enzyme or anti-protein antibody comprising administering to a non-human animal a nucleic acid as set forth in claim 1 or claim 57 or a subsequence thereof in an amount sufficient to generate a humoral immune response, thereby making an anti-enzyme or anti-protein antibody.
146. A method of making an anti-enzyme or anti-protein antibody comprising administering to a non-human animal a polypeptide or peptide as set forth in claim 76, or OCH-tϋ-ZW lHU.tU/ UZZ /U-I WU
a subsequence thereof, in an amount sufficient to generate a humoral immune response, thereby making an anti-enzyme or anti-protein antibody.
147. A method of producing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid operably linked to a promoter, wherein the nucleic acid comprises a sequence as set forth in claim 1 or claim 57; and (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide.
148. The method of claim 147, further comprising transfoπning a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide.
149. A method for identifying a polypeptide having an enzyme activity or encoding a protein comprising the following steps:
(a) providing a polypeptide as set forth in claim 76;
(b) providing an enzyme substrate; and
(c) contacting the polypeptide with the substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of a reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having an enzyme activity or encoding a protein.
150. A method for identifying an enzyme or protein substrate comprising the following steps: (a) providing a polypeptide as set forth in claim 76;
(b) providing a test substrate; and
(c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting a decrease in the amount of substrate or an increase in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of a reaction product identifies the test substrate as an enzyme or protein substrate.
151. A method of determining whether a test compound specifically binds to a polypeptide comprising the following steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid has a sequence as set forth in claim 1 or claim 57;
(b) providing a test compound; (c) contacting the polypeptide with the test compound; and
(d) determining whether the test compound of step (b) specifically binds to the polypeptide.
152. A method of determining whether a test compound specifically binds to a polypeptide comprising the following steps:
(a) providing a polypeptide as set forth in claim 76;
(b) providing a test compound;
(c) contacting the polypeptide with the test compound; and
(d) determining whether the test compound of step (b) specifically binds to the polypeptide.
153. A method for identifying a modulator of an enzyme activity comprising the following steps:
(a) providing a polypeptide as set forth in claim 76; (b) providing a test compound;
(c) contacting the polypeptide of step (a) with the test compound of step (b) and measuring an activity of the enzyme, wherein a change in the enzyme activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the enzyme activity.
154. The method of claim 153, wherein the enzyme activity is measured by providing an enzyme substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product, or, an increase in the amount of the substrate or a decrease in the amount of a reaction product, and optionally a decrease in the amount of the substrate or an increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of an enzyme activity, and optionally an increase in the amount of the substrate or a decrease in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of an enzyme activity.
155. A computer system comprising a processor and a data storage device wherein said data storage device has stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises sequence as set forth in claim 76, a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57.
156. The computer system of claim 155, further comprising a sequence comparison algorithm and a data storage device having at least one reference sequence stored thereon, and optionally the sequence comparison algorithm comprises a computer program that indicates polymorphisms, and optionally the computer system further comprises an identifier that identifies one or more features in said sequence.
157. A computer readable medium having stored thereon a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide as set forth in claim 76, a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57.
158. A method for identifying a feature in a sequence comprising the steps of: (a) reading the sequence using a computer program which identifies one or more features in a sequence, wherein the sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide as set forth in claim 76, a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57; and (b) identifying one or more features in the sequence with the computer program.
159. A method for comparing a first sequence to a second sequence comprising the steps of: (a) reading the first sequence and the second sequence through use of a computer program which compares sequences, wherein the first sequence comprises a polypeptide sequence or a nucleic acid sequence, wherein the polypeptide sequence comprises a polypeptide as set forth in claim 76, a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57; and (b) determining differences between the first sequence and the second sequence with the computer program, and optionally the step of determining differences between the first sequence and the second sequence further comprises the step of identifying polymorphisms, and optionally the method further comprises use of an identifier that identifies one or more features in a sequence and optionally the method further comprises reading the first sequence using a computer program and identifying one or more features in the sequence.
160. A method for isolating or recovering a nucleic acid encoding a polypeptide with an enzyme activity or encoding a protein from an environmental sample comprising the steps of:
(a) providing an amplification primer pair as set forth in any of claims 54 to 56; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to the amplification primer pair; and,
(c) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the environmental sample, thereby isolating or recovering a nucleic acid encoding a polypeptide with an enzyme activity or encoding a protein from an environmental sample; and optionally each member of the amplification primer sequence pair comprises an oligonucleotide comprising at least about 10 to about 50 consecutive bases of a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO: 1 through SEQ ID NO: 193, or a subsequence thereof.
161. A method for isolating or recovering a nucleic acid encoding a polypeptide with an enzyme activity or encoding a protein from an environmental sample comprising the steps of:
(a) providing a polynucleotide probe comprising a sequence as set forth in claim 1 or claim 57, or a subsequence thereof; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a);
(c) combining the isolated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (a); and
(d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide with an enzyme activity or encoding a protein from an environmental sample.
162. The method of claim 160 or claim 161, wherein the environmental sample comprises a water sample, a liquid sample, a soil sample, an air sample or a biological sample, and optionally the biological sample is derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell.
163. A method of generating a variant of a nucleic acid encoding a polypeptide with an enzyme activity or encoding a protein comprising the steps of:
(a) providing a template nucleic acid comprising a sequence as set forth in claim 1 or claim 57; and
(b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid, and optionally the method further comprises expressing the variant nucleic acid to generate a variant enzyme or protein polypeptide.
164. The method of claim 163, wherein the modifications, additions or deletions are introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a combination thereof, or, the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection ijCK+to-zu ltυ.tu/ UΔL /u-i w u
mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
165. The method of claim 163, wherein the method is iteratively repeated until an enzyme or protein having an altered or different activity or an altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced, and optionally the variant enzyme or protein polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature, or the variant enzyme or protein polypeptide has increased glycosylation as compared to the enzyme or protein encoded by a template nucleic acid, or the variant enzyme or protein polypeptide has an enzyme activity under a high temperature, wherein the enzyme encoded by the template nucleic acid is not active under the high temperature and optionally the method is iteratively repeated until an enzyme or protein coding sequence having an altered codon usage from that of the template nucleic acid is produced, or the method is iteratively repeated until an enzyme or protein gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.
166. A method for modifying codons in a nucleic acid encoding a polypeptide with an enzyme activity or encoding a protein to increase its expression in a host cell, the method comprising the following steps:
(a) providing a nucleic acid encoding a polypeptide with an enzyme activity or encoding a protein comprising a sequence as set forth in claim 1 or claim 57; and,
(b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
167. A method for modifying codons in a nucleic acid encoding a polypeptide having an enzyme activity or encoding a protein, the method comprising the following steps: juttυ-iu iny.tυ/ u^i / υ- i vv υ
(a) providing a nucleic acid encoding a polypeptide with an enzyme activity or encoding a protein comprising a sequence as set forth in claim 1 or claim 57; and,
(b) identifying a codon in the nucleic acid of step (a) and replacing it with a different codon encoding the same amino acid as the replaced codon, thereby modifying codons in a nucleic acid encoding an enzyme or a protein.
168. A method for modifying codons in a nucleic acid encoding a polypeptide having an enzyme activity or encoding a protein, to increase its expression in a host cell, the method comprising the following steps: (a) providing a nucleic acid encoding a polypeptide having an enzyme activity or encoding a protein, comprising a sequence as set forth in claim 1 or claim 57; and,
(b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.
169. A method for modifying a codon in a nucleic acid encoding a polypeptide having an enzyme activity or encoding a protein to decrease its expression in a host cell, the method comprising the following steps:
(a) providing a nucleic acid encoding a polypeptide having an enzyme activity or encoding a protein comprising a sequence as set forth in claim 1 or claim 57; and
(b) identifying at least one preferred codon in the nucleic acid of step (a) and replacing it with a non-preferred or less preferred codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in a host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to decrease its expression in a host cell, and optionally the host cell is a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or a mammalian cell.
170. A method for producing a library of nucleic acids encoding a plurality of modified enzyme or protein active sites or substrate binding sites, wherein the modified active sites or substrate binding sites are derived from a first nucleic acid comprising a sequence encoding a first active site or a first substrate binding site the method comprising the following steps:
(a) providing a first nucleic acid encoding a first active site or first substrate binding site, wherein the first nucleic acid sequence comprises a sequence that hybridizes under stringent conditions to a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO: 1 through SEQ ID NO: 193, or a subsequence thereof, and the nucleic acid encodes an enzyme or protein active site or an enzyme or protein substrate binding site;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of active site- encoding or substrate binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified enzyme or protein active sites or substrate binding sites.
171. The method of claim 170, comprising mutagenizing the first nucleic acid of step (a) or variants by a method comprising an optimized directed evolution system, Gene Site Saturation Mutagenesis (GSSM), or a synthetic ligation reassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, and a combination thereof; or, mutagenizing the first nucleic acid of step (a) or variants by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.
172. A method for making a small molecule comprising the following steps:
(a) providing a plurality of biosynthetic enzymes capable of synthesizing or modifying a small molecule, wherein one of the enzymes comprises an enzyme encoded by a nucleic acid comprising a sequence as set forth in claim 1 or claim 57;
(b) providing a substrate for at least one of the enzymes of step (a); and
(c) reacting the substrate of step (b) with the enzymes under conditions that facilitate a plurality of biocatalytic reactions to generate a small molecule by a series of biocatalytic reactions.
173. A method for modifying a small molecule comprising the following steps: (a) providing an enzyme, wherein the enzyme comprises a polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence as set forth in claim 1 or claim 57; (b) providing a small molecule; and
(c) reacting the enzyme of step (a) with the small molecule of step (b) under conditions that facilitate an enzymatic reaction catalyzed by the enzyme, thereby modifying a small molecule by an enzymatic reaction, and optionally the method further comprises use of a plurality of small molecule substrates for the enzyme of step (a), thereby generating a library of modified small molecules produced by at least one enzymatic reaction catalyzed by the enzyme, and optionally the method further comprises use of a plurality of additional enzymes under conditions that facilitate a plurality of biocatalytic reactions by the enzymes to form a library of modified small molecules produced by the plurality of enzymatic reactions, and optionally the method further comprises the step of testing the library to determine if a particular modified small molecule which exhibits a desired activity is present within the library, and optionally the step of testing the library further comprises the steps of systematically eliminating all but one of the biocatalytic reactions used to produce a portion of the plurality of the modified small molecules within the library by testing the portion of the modified small molecule for the presence or absence of the particular modified small molecule with a desired activity, and identifying at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.
174. A method for determining a functional fragment of an enzyme comprising the steps of:
(a) providing an enzyme, wherein the enzyme comprises a polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence as set forth in claim 1 or claim 57; and
(b) deleting a plurality of amino acid residues from the sequence of step (a) and testing the remaining subsequence for an enzyme activity, thereby determining a functional fragment of an enzyme, and optionally the enzyme activity is measured by providing an enzyme substrate and detecting a decrease in the amount of the substrate or an increase in the amount of a reaction product.
175. A method for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps:
(a) making a modified cell by modifying the genetic composition of a cell, wherein the genetic composition is modified by addition to the cell of a nucleic acid comprising a sequence as set forth in claim 1 or claim 57;
(b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and,
(d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis, and optionally the genetic composition of the cell is modified by a method comprising deletion of a sequence or modification of a sequence in the cell, or, knocking out the expression of a gene, and optionally the method further comprises selecting a cell comprising a newly engineered phenotype, and optionally the method further comprises culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.
176. An isolated or recombinant signal sequence (signal peptide) consisting of, or consisting essentially of, a sequence as set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46, or 1 to 47, of
(a) SEQ ID NO:2, SEQ ID NO.4, SEQ ID NO6, SEQ ID NO.8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO.16, SEQ ID NO: 18, SEQ ID NO 20, SEQ
ID NO:22, SEQ ID NO.24, and all polypeptides disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO: 194, or
(b) a sequence as set forth in claim 1 or claim 57.
177. A chimeric polypeptide comprising at least a first domain comprising signal peptide (SP) having a sequence as set forth in claim 176, and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP), and optionally the heterologous polypeptide or peptide is not an enzyme, and optionally the heterologous polypeptide or peptide is amino terminal to, carboxy terminal to or on both ends of the signal peptide (SP) or an enzyme catalytic domain (CD).
178. A method of increasing thermotolerance or thermostability of a enzyme polypeptide, the method comprising glycosylating an enzyme, wherein the polypeptide comprises at least thirty contiguous ammo acids of a polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, thereby increasing thermotolerance or thermostability of the enzyme.
179 A method for overexpressing a recombinant enzyme in a cell compπsmg expressing a vector comprising a nucleic acid sequence as set forth in claim 1 or claim 57, wherein overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector
180. A method of making a transgenic plant composing the following steps:
(a) introducing a heterologous nucleic acid sequence into the cell, wherein the heterologous nucleic sequence compπses a sequence as set forth in claim 1 or claim 57, thereby producing a transformed plant cell,
(b) producing a transgenic plant from the transformed cell, and optionally step (a) further comprises introducing the heterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts, and optionally step (a) comprises introducing the heterologous nucleic acid sequence directly to plant tissue by DNA particle bombardment or by using an Agrobacterium tumefaciens host.
181. A method of expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps:
(a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence comprises a sequence as set forth in claim 1 or claim 57;
(b) growing the plant under conditions wherein the heterologous nucleic acids sequence is expressed in the plant cell.
182. A method for decreasing the amount of a compound in a composition comprising the following steps:
(a) providing a polypeptide having an enzyme activity or encoding a protein as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57; (b) providing a composition comprising the compound; and
(c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the enzyme hydrolyzes, dehalogenates, oxidizes, breaks up or otherwise processes the compound in the composition.
183. A cellulose-comprising compound comprising at least one polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, and optionally the cellulose-comprising compound comprises a pulp, a paper, a paper product, a wood, a wood product, or a paper or wood waste product.
184. A method for chemical bleaching of lignin or a cellulose in a pulp or paper manufacturing process comprising the steps of (a) providing a polypeptide having chloroperoxidase activity as set forth in claim 76, or a polypeptide having chloroperoxidase activity encoded by a nucleic acid as set forth in claim 1 or claim 57;
(b) providing a compound comprising a lignin or a cellulose; and (c) contacting the compound with the polypeptide under conditions wherein the polypeptide is enzymatically active and the lignin or a cellulose is bleached, wherein optionally the enzymatic activity comprises a heme-based peroxidase activity, and optionally the enzyme having heme-based peroxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO:21 ; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO: 39; SEQ ID NO:41 ; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO: 49 or SEQ ID NO:51 , or the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively, and optionally the enzyme has a heme-based chloroperoxidase activity and is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1, or the enzyme has an amino acid sequence as set forth in SEQ ID NO:2, wherein optionally the enzymatic activity comprises a non-heme-based peroxidase activity, and optionally the enzyme having non-heme-based chloroperoxidase activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 ; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively, and optionally the cellulose or lipophilic compound comprises a pulp, a paper, a paper product, a wood, a wood product, or a paper or wood waste product.
185. A composition comprising at least one polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, wherein optionally the composition is formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a pill, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof, and optionally the composition comprises at least two or three polypeptides as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
15%, 20% or 25%.
186. A pharmaceutical composition comprising a polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, wherein optionally the pharmaceutical composition is formulated as an edible delivery agent, an injectable liquid, a spray, a tablet, a gel, a liposome, a capsule, a geltab, a hydrogel, a lotion, a topical applied liquid, a suppository, an aerosol, a powder, a lyophilized compound, a propellant, a foam, an emulsion, a nanostructure, an implant or a combination thereof wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%%.
187. A nanostructure comprising at least one polypeptide as set forth in claim
76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, wherein optionally the nanostructure comprises a nanotubule or a nanofiber, and optionally the composition comprises at least two or three polypeptides as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57.
188. A lyophilized polypeptide having a sequence as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57.
189. A decontaminating, neutralizing or detoxifying composition comprising at least one polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, wherein optionally the decontaminating or detoxifying composition is formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof, wherein optionally the composition comprises at least two or three polypeptides as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, wherein optionally an enzyme in the composition is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
190. The decontaminating, neutralizing or detoxifying composition of claim 189, further comprising a surfactant, an emulsifier, a foaming agent or a combination thereof.
191. The decontaminating, neutralizing or detoxifying composition of claim
189, wherein the composition is formulated as a pesticide, herbicide and/or insecticide detoxifying agent.
192. The decontaminating, neutralizing or detoxifying composition of claim 189, wherein the composition is formulated as a nerve gas detoxifying agent.
193. The decontaminating, neutralizing or detoxifying composition of claim
189, wherein when the composition comprises a haloperoxidase enzyme, the composition also comprises halite component, and optionally the halite component comprises a chlorite component, and optionally the chlorite component comprises a sodium chlorite or a sodium iodite.
194. A product of manufacture comprising at least one polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57.
195 The product of manufacture of claim 194, wherein when the product of manufacture composes a haloperoxidase enzyme, the composition also comprises halite component, and optionally the halite component compnses a chlorite component, and optionally the chlonte component compnses a sodium chlonte or a sodium iodite
196 A cloth, textile or fiber compπsmg at least one polypeptide as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57, wherem optionally the at least one polypeptide is immobilized onto the surface of the cloth, textile or fiber, or the at least one polypeptide is a component of a coatmg or covering on the surface of the cloth, textile or fiber, or a formulation embedded or washed into or onto the cloth, textile or fiber, wherein optionally the composition compnses at least two or three polypeptides as set forth in claim 76, or a polypeptide encoded by a nucleic acid as set forth in claim 1 or claim 57,
197 The decontaminating, neutralizing or detoxifying composition of claim 189, compnsing a mixture of at least two, three, four, five or six different classes of enzymes, wherein optionally each class of enzyme detoxifies a different toxic agent or biological agent
198 The decontaminating, neutralizing or detoxifying composition of claim
197, compnsing a mixture of at least three different classes of enzymes compnsmg at least one dehalogenase, at least one haloperoxidase, and at least one organophosphonc acid anhydrolase (OPAA), and optionally the haloperoxidase is a chloroperoxidase
199 The decontaminating, neutralizing or detoxifying composition of claim
198, wherem (a) the dehalogenase is a polypeptide having a sequence as set forth in SEQ ID NO 69 or SEQ ID NO 91 , or has an amino acid sequence as set forth in SEQ ID
NO 70 or SEQ ID NO 92, respectively, or (b) the chloroperoxidase is a heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO 1 , or having an ammo acid sequence as set forth in SEQ ID NO 2, or (c) the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51 , or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID
NO:50 or SEQ ID NO:52, respectively; or (d) the haloperoxidase is a non-heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 ; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively; or (e) the organophosphoric acid anhydrolase (OPAA) is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having a sequence as set forth in SEQ ID NO: 194.
200. The decontaminating, neutralizing or detoxifying composition of claim 197, comprising a mixture of at least two different classes of enzymes comprising at least one diisopropylfluorophosphatase (DFPase) or organophosphoric acid anhydrolase (OPAA), and at least one haloperoxidase, and optionally the haloperoxidase is a chloroperoxidase.
201. The decontaminating, neutralizing or detoxifying composition of claim 200, comprising a diisopropylfluorophosphatase (DFPase), a organophosphoric acid anhydrolase (OPAA) and a haloperoxidase.
202. The decontaminating, neutralizing or detoxifying composition of claim 200, wherein the DFPase and/or OPAA are used to decontaminate, neutralize or detoxify G agents, and the haloperoxidase or chloroperoxidase (CPO) are used to decontaminate, ocw+o-zu itυ.tυ/ u/z /w-i wυ
neutralize or detoxify V agents, H agents and/or biological agents, wherein optionally the biological agent is a bacterial spore.
203. The decontaminating, neutralizing or detoxifying composition of claim 200, further comprising a dehalogenase.
204. The decontaminating, neutralizing or detoxifying composition of claim 200, further comprising a cholinesterase.
205. The decontaminating, neutralizing or detoxifying composition of claim
200, wherein the diisopropyl-fluorophosphatase (DFPase) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72.
206. The decontaminating, neutralizing or detoxifying composition of claim
200, wherein the haloperoxidase is a chloroperoxidase.
207. The decontaminating, neutralizing or detoxifying composition of claim 206, wherein the chloroperoxidase is a heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1, or having an amino acid sequence as set forth in SEQ ID NO:2; or the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO: 13; SEQ ID NO.15; SEQ ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO.23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31 ; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO.39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO.6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO: 50 or SEQ ID NO: 52, respectively; or the haloperoxidase is a non-heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 ; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
208. The decontaminating, neutralizing or detoxifying composition of claim 200, wherein the organophosphoric acid anhydrolase (OPAA) activity is encoded by a nucleic having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
209. The decontaminating, neutralizing or detoxifying composition of claim 197, comprising a mixture of at least two different classes of enzymes comprising at least one dehalogenase (DH) and at least one haloperoxidase, wherein optionally the haloperoxidase is a chloroperoxidase (CPO).
210. The decontaminating, neutralizing or detoxifying composition of claim 209, wherein the dehalogenases and haloperoxidases are used to decontaminate, neutralize or detoxify H agents.
211. The decontaminating, neutralizing or detoxifying composition of claim
209, wherein the haloperoxidase is a heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO: 1 , or having an amino acid sequence as set forth in SEQ ID NO:2; or the haloperoxidase is a heme-based peroxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID NO:17; SEQ ID NO: 19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:45; SEQ ID NO:47; SEQ ID NO:49 or SEQ ID NO:51, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50 or SEQ ID NO:52, respectively; jutτw-.ώv/ 1 -τu.-iΛj/ i^z.ώ / υ- 1 vv vy
or the haloperoxidase is a non-heme-based chloroperoxidase encoded by a nucleic having a sequence as set forth in SEQ ID NO:1; SEQ ID NO:53; SEQ ID NO:55; SEQ ID NO:57; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65 or SEQ ID NO:67, or, the enzyme has an amino acid sequence as set forth in SEQ ID NO:2; SEQ ID 5 NO:54; SEQ ID NO:56; SEQ ID NO:58; SEQ ID NO:60; SEQ ID NO:62; SEQ ID NO:64; SEQ ID NO:66 or SEQ ID NO:68, respectively.
212. The decontaminating, neutralizing or detoxifying composition of claim 209, wherein the dehalogenase has a sequence as set forth in SEQ ID NO:69 or SEQ ID o NO:91 , or has an amino acid sequence as set forth in SEQ ID NO:70 or SEQ ID NO.92, respectively.
213. The decontaminating, neutralizing or detoxifying composition of claim 209, further comprising adding at least one diisopropylfluorophosphatase (DFPase),5 organophosphoric acid anhydrolase (OPAA) and/or cholinesterase to decontaminate, neutralize or detoxify G agents.
214. The decontaminating, neutralizing or detoxifying composition of claim 213, wherein the diisopropyl-fluorophosphatase (DFPase) is encoded by a nucleic acid0 having a sequence as set forth in SEQ ID NO:71, or has an amino acid sequence as set forth in SEQ ID NO:72.
215. The decontaminating, neutralizing or detoxifying composition of claim 213, wherein the organophosphoric acid anhydrolase (OPAA) activity is encoded by a5 nucleic having a sequence as set forth in SEQ ID NO: 193, or having an amino acid sequence as set forth in SEQ ID NO: 194.
216. The decontaminating, neutralizing or detoxifying composition of claim 197, comprising a mixture of at least two different classes of enzymes comprising at least0 one dehalogenase (DH) enzyme and at least one organophosphoric acid anhydrolase (OPAA).
217. The decontaminating, neutralizing or detoxifying composition of claim 216, wherein the dehalogenases is used to decontaminate, neutralize or detoxify an H agent, and the organophosphoric acid anhydrolase is used to decontaminate, neutralize or detoxify G agents.
218. The decontaminating, neutralizing or detoxifying composition of claim 216, further comprising at least one haloperoxidase to augment the decontamination, neutralization, detoxification of H agents.
219. The decontaminating, neutralizing or detoxifying composition of claim 216, further comprising a haloperoxidase, and optionally the composition can also comprise a halite component, wherein optionally the halite component comprises an iodite or a chlorite component; and optionally the iodite or a chlorite component comprises a sodium chlorite or a sodium iodite or equivalent components.
220. The decontaminating, neutralizing or detoxifying composition of claim 216, further comprising at least one diisopropylfluorophosphatase (DFPase) and/or cholinesterase enzyme to supplement the OPAA decontamination, neutralization or detoxification of a G agent.
221. A foaming agent comprising a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221 , wherein optionally an enzyme in the foaming agent is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
222. An emulsifying agent or a surfactant comprising a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, wherein optionally an enzyme in the foaming agent or surfactant is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
223. A paint or coating comprising a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, wherein optionally an enzyme in the paint or coating is present at a concentration of about 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%, or is present at a w/v of about 0.25%, 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or 25%.
224. A method for decontaminating, neutralizing or detoxifying a toxic agent comprising application or administration of a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, or a pharmaceutical composition as set forth in claim 186.
225. The method of claim 224, wherein the composition is formulated as an edible delivery agent, an injectable liquid, a tablet, a gel, a liposome, a capsule, a geltab, a lotion, a topical applied liquid, a suppository, a powder, a lyophilized compound, a foam, an emulsion or a combination thereof.
226. A method for preventing the toxic effects of a V agent, an H agent, a G agent or a biological agent comprising application or administration of a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, or a pharmaceutical composition as set forth in claim 186.
227. A gas mask comprising a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, or a pharmaceutical composition as set forth in claim 186.
228. An air or water filter comprising a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, or a pharmaceutical composition as set forth in claim 186.
229. A pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agent comprising a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, or a pharmaceutical composition as set forth in claim 186, wherein optionally an enzyme in the composition is present at a concentration of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or is present at a w/v of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%.
230. The pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agent of claim 229, wherein the composition comprises an organophosphoesterase that hydrolyzes P-S or P-F bonds.
231. The pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agent of claim 230, wherein the organophosphoesterase acts as an inhibitor of an acetyl-cholinesterases or a butyrylcholinesterase.
232. The pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agent of claim 229, wherein the pesticide is Demeton-S, Demeton-S- methyl, Demeton-S-methylsulphon, Demeton-methyl, Parathion, Phosmet,
Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos-ethyl, Amiton, Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton, Endothion, Etion, Ethoate- methyl, Formothion, Malathion, Mercarbam, Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone, Prothidathion, Prothoate, Sophamide, Thiometon, Vamidothion, Methamidophos or a combination thereof.
233. The pesticide, herbicide and/or insecticide decontaminating, neutralizing or detoxifying agent of claim 229, wherein the composition is formulated as or with a coating, a paint, a foam, a liquid, a gel, a lotion, a surfactant, a powder or an emulsifier.
234. A method for decontaminating, neutralizing or detoxifying a pesticide, herbicide and/or insecticide comprising application or administration of a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, or a pharmaceutical composition as set forth in claim 186, or the pesticide decontaminating, neutralizing or detoxifying agent of any of claims 228 to 232.
235. A method for preventing the toxic effects of a pesticide, herbicide and/or insecticide comprising application or administration of a decontaminating, neutralizing or detoxifying composition of any one of claims 189 to 193 or any one of claims 197 to 221, or a pharmaceutical composition as set forth in claim 186, or the pesticide decontaminating, neutralizing or detoxifying agent of any of claims 228 to 232.
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