CA2208223A1 - Fusarium isolate and lipases, cutinases and enzyme compositions derived therefrom - Google Patents
Fusarium isolate and lipases, cutinases and enzyme compositions derived therefromInfo
- Publication number
- CA2208223A1 CA2208223A1 CA002208223A CA2208223A CA2208223A1 CA 2208223 A1 CA2208223 A1 CA 2208223A1 CA 002208223 A CA002208223 A CA 002208223A CA 2208223 A CA2208223 A CA 2208223A CA 2208223 A1 CA2208223 A1 CA 2208223A1
- Authority
- CA
- Canada
- Prior art keywords
- lipases
- lipolytic enzymes
- lipase
- fusarium
- kda
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- 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)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- 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)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
- Y10S435/925—Cephalosporium
Abstract
The present invention concerns lipolytic enzymes naturally produced by a fungus of the species Fusarium solanii. The lipolytic enzymes include two lipases and a cutinase. The present invention concerns a biologically pure culture of Fusarium solanii producing these lipolytic enzymes and in particular a biologically pure culture of Fusarium solanii var. minus T.92.637/1. The present invention concerns also a detergent composition containing these lipolytic enzymes.
Description
WO 96/18729 PCT/EP9~i/04799 Fusarium isolate and lipases, cutinases and enzyme compositions derived therefrom The present invention relates to a novel, biologically pure culture of an isolate of the genus Fusarium, lipolytic enzymes, lipases, cl-tin~es and enzyme compositions derived the-crl.~m and, in particular, to a biologically pure culture of Fusarium solanii var. minus T.92.637/1, lipolytic enzymes, S lipases, cutin~es and enzyme compositions derived th~-~r ol.l and the use of such enzymes in d~;lel~el-L compositions.
Lipolytic enzymes, including lipases and cutin~ces, are commonly employed in dele.~ent cleaning compositions for the removal of fatty acid-based dirt and stains. As such, they have to be stable in presence of 10 these d~telgellLs~ and in particular in ~lk~line detergent solutions. Further, lipolytic enzymes are often used in combination with other enzymes, such as ~lk~lin~ proteases, amylases, oxidases and/or other proteins, thereby requiring them to also be stable in presence of these enzymes.
Lipolytic enzymes n~tllr~lly produced by various fungi are known.
Eu~ean Patent Application 0 130 064 describes an enzymatic d~;~e.gent additive including a lipase n~tllr~lly produced by the fungal strain Fusarium oxysporum.
Tnt~rn~tional Patent Application WO 90/09446 discloses an enzymatic dele.gt;ll~ composition which incllldes a cutin~e naturally produced by 20 Fusarium solanii var. pisi.
To the best of our knowledge, we are not aware of any other lipolytic enzyme naturally produced by any other strains of Fusarium. Further, we are not aware of either any lipase or cutinase which has been n~tllr~lly produced by any other species of Fusarium, including any isolate of the 25 species Fusarium solanii var. minus.
It is a prirnary object of the present invention is to provide novel lipolytic enzymes naturally produced by a fungus of the species Fusarium solanii. By the term "lipolytic enzyme" (E.C. 3.1.1), what is meant herein is a lipase (E.C. 3.1.1.3) or a cutinase (E.C. 3.1.1.50) which is capable of 30 removing stains of a fatty nature. Lipase has higher selectivity toward long chain triglycerides contained in fat than cutinase. Cutinase has higher selectivity toward short chain triglycerides contained in fat than lipase.
It is a primary object of the present invention to provide novel lipases which, when incorporated in a deLergelll composition, are capable of removing fatty acid based dirt and stains from fabrics.
It is another primary object of the present invention to provide a novel cutinase which, when incorporated in a delel~enL composition, is capable of removing fatty acid based dirt and stains from fabrics.
Sti another primary object of the present invention is to provide novel enzyme compositions of lipases and/or clltin~ces which, when incorporated in a detergent composition, are capable of removing fatty acid based dirt and stains from fabrics.
A further primary object of the present invention is to identify, isolate and provide a biologically pure culture of a novel fungus of the genus Fusarium which is capable of naturally producing the lipolytic enzymes, including the lipases and cutinases, of the present invention which lipolytic enzymes may be incorporated into the enzymatic compositions and/or the d~ler~enl compositions of the present invention.
A yet further primary object of the present invention is to provide a method for removing fatty acid-based dirt and stains from fabrics with the use of the enzymes and/or the enzyme compositions of the present mventlon.
A still yet further object of the present invention is to provide an enzymatic d~;~elgent composition capable of removing fatty acid-based dirt and stains from fabrics, which dele.~enl composition includes a lipolytic enzyme and/or a lipase and/or cutin~e and/or enzyme composition.
In accordance with the te~hing~ of the present invention, disclosed herein is a biologically pure culture of the novel isolate F~ rillm solanii var. minus T.92.637/1 and ~ c and derivatives thereof. This isolate is capable of producing the lipolytic enzymes, the lipases, the clltin~es and the enzyme compositions of the present invention.
In another aspect of the present invention, disclosed herein are novel lipolytic enzymes which are naturally produced by an isolate of the species Fusarium and, more particularly, of the species Fusarium solanii. The ~cr~illed enzymes are those lipolytic enzymes which are naturaUy produced by the isolate Fusarium solanii var. minus. The most p-cre--~d enzymes are those lipolytic enzymes which are naturally produced by the isolate Fusarium solanii var. minus T.92.637/1.
In one pl~rerled embodiment, the lipolytic enzymes of the present invention include a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa. In another pl~f~lled S embodiment, the lipolytic enzymes include a lipase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 lcDa.
In still another preferred embodiment, the lipolytic enzymes include a cntin~e having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa.
In a yet further aspect of the present invention, disclosed herein are novel enzyme compositions having at least one lipolytic enzyme which is n~ lly produced by a fungus of the species Fusarium and, more particularly, from the species Fusarium solanii. Most plerell~;d are those enzyme compositions having at least one lipolytic enzyme which is n~t~ lly produced by the isolate Fusarium solaniu var. minus T.92.637/1.
In a ~lc;r~lled embodiment, the lipolytic enzyme of the enzyme compositions is a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa. Alternatively, the lipolytic enzyme of the enzyme compositions is a lipase having an isoelectric point of 20 about 5.2 and/or an a~al~ molecular weight of about 60 IcDa. Still further alLellld~ ely, the lipolytic enzyme of the enzyme compositions is a c~-tin~e having an isoelectric point of about 7.2 and/or an a~are;lll molecular weight of about 22 kDa.
In a further ~lcfell~d embodiment, the enzyme compositions contain at 25 least two of the aforesaid lipolytic enzymes. In one ~ relled embodiment, the lipolytic enzymes are a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a lipase having an isoelectric point of about 5.2 and/or an a~par~nl molecular weight of about 60 kDa. In an alternative plerelr~d embodiment, the lipolytic enzymes are 30 a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a cutinase a having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa.
In still another alternative pl~rell~d embodiment, the lipolytic enzymes are a lipase having an isoelectric point of about 5.2 and/or an a~l~altl~t 35 molecular weight of about 60 kDa and a cutinase having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa.
Most pl~re..ed are those enzyme compositions that contain all three of the lipolytic enzymes Such compositions have a lipase having an isoelectric point of about 6 9 and/or an apparent molecular weight of about 30 kDa and a lipase having an isoelectric point of about 5 2 and/or an S apparent molecular weight of about 60 kDa and a c~ tin~e having an isoelectric point of about 7 2 and/or an apparent molecular weight of about 22 kDa In a still yet further aspect of the present invention, disclosed herein are novel enzymatic d~le.~t;--~ compositions having at least one lipolytic enzyme 10 which is n~hlr~lly produced by a fungus of the species Fusarium and, more particularly, from the species Fusarium solanii Most ~ fe-led are those enzymatic deler~en~ compositions having at least one lipolytic enzyme which is naturally produced by the isolate Fusarium solanii var minus T 92.637/1 In a ~.ere..~d embodiment, the lipolytic enzyme of the enzymatic 15 delerge..l compositions is a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa Alternatively, the lipolytic enzyme of the enzymatic d~ compositions is a lipase having an isoelectric point of about 5 2 and/or an apparent molecular weight of about 60 kDa Still further alternatively, Ihe lipolytic enzyme of the 20 enzymatic d~e-~--L compositions is a clltin~e having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa In a further ~lc;relled embodiment, the enzymatic d~
compositions contain at least two of the aforesaid lipolytic enzymes. In one p-er~ d embodiment, the lipolytic enzymes are a lipase having an 25 isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a lipase having an isoelectric point of about 5 2 and/or an a~pdl~ell~ molecular weight of about 60 kDa In an alternative ~ ;;rellcd embodiment, the lipolytic enzymes are a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a 30 clltin~e a having an isoelectric point of about 7 2 and/or an apparent molecular weight of about 22 kDa. In still another alternative ~.~rell~d embodiment, the lipolytic enzymes are a lipase having an isoelectric point of about 5 2 and/or an apparent molecular weight of about 60 kDa and a cutinase a having an isoelectric point of about 7 2 and/or an apparent 35 molecular weight of about 22 kDa Most p-efe--~d are those enzymatic detergent compositions that contain all three of the lipolytic enzymes. Such compositions have a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a lipase having an isoelectric point of about 5.2 and/or an a~e.lL molecular weight of about 60 kDa and a cutinase having an S isoelectric point of about 7.2 and/or an a~en~ molecular weight of about 22 kDa.
The enzymatic detergent compositions of the present invention are all capable of removing fatty acid-based dirt and stains from fabrics.
These and further objects and advantages of the present invention will become readily apparent from a reading of the following description.
The novel isolate of the present invention is a biologically pure culture of the isolate Fusarium solanii var. minus T.92.637/1.
The fungus Fusarium solanii var. minus T.92.637/1 was deposited under the provisions of the Budapest Treaty on 23 November 1993 with the BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS
(MUCL: THE AGRICULTURAL AND INDUSTRIAL FILAMENTOUS
AND YEAST FUNGI COLLECTION, Mycothéque de l'Université
Catholique de Louvain, Place Croix du Sud 3, B-1348 Louvain-La-Neuve, Belgium under accession number MUCL 38667).
The morphological characteristics of Fusarium solanii var. minus T.92.637/1 are set forth in detail below in Example 1.
Natural and artificial mllt~ntc and derivatives of Fusarium solanii var.
minus T.92.637/1 may be obtained by natural modifications or by genetic morlifi~tions thereof. Such well known methods as, for example, by X-ray techniques, ultraviolet irradiation, chemical mutagens or genetic engineering can all be employed to obtain such mllt~ntC and derivatives. It is to be expressly understood that such mutants and derivatives are also contemplated and encomp~csed by the present invention.
The novel lipolytic enzymes, lipases, cutinases and enzyme compositions of the present invention are n~t~ lly produced by a fungus of the genus Fusarium and, more particularly, from the isolate Fusarium solanii var. minus T.92.637/1.
The lipolytic enzymes, lipases, c~ltin~ces and enzyme compositions of the present invention are naturally produced during the growth of the fungus and are subst~nti~lly extracellularly excreted (secreted) into the medium.
These lipolytic enzymes, lipases, cutinases and enzyme compositions may be recovered using normal techni(lues, generally comprising centrifugation to remove the fungal cells, followed by concentration by ultraf~tration. The crude lipolytic enzymes, lipases, cutin~ces and enzyme compositions obtained in this way can then be dried. An additional purific~tion stage is also possible in order to separate cont~min~tin~ proteins from the lipolytic enzymes, lipases, cutin~ces and enzyme compositions of the present nvention.
One of the lipases of the present invention is characterized by having an isoelectric point of about 5.2 and an apparent molecular weight of about 60 kDa. Another of the lipases of the present invention is characterized by having an isoelectric point of about 6.9 and an apparent molecular weight of about 30 kDa.
The cutinase of the present invention is characterized by having an isoelectric point of about 7.2 and an appalel.~ molecular weight of about 22kD.
The enzyme compositions of the present invention are comprised of the lipolytic enzymes and/or lipases and/or cutin~ces of the present invention.
More particularly, the enzyme compositions include either at least one lipase and/or at least one cutin~ce naturally produced by Fusarium solanii var. minus. T.92.637/1. In a pl~relled embodiment, the enzyme composition of the present invention includes at least one of the lipases and at least one of the cutinases naturally produced by Fusarium solanii var.
minus. T.92.637/1. In another embo-liment, the enzyme composition incllldes the lipases which are naturally produced by Fusarium solanii var.
minus. T.92.637/1. Most ~l~;Çt;lled, is that enzyme composition includes the lipases and the cutin~ces naturally produced by Fusarium solanii var.
minus. T.92.637/1.
The lipolytic enzymes (lipases and cutinases) and enzyme compositions of the present invention have use in a number of varying applications for hydrolyzing and modifying fats, ranging from, inter alia, oleochemistry processing applications to soap and dt;ler~en~ compositions. For example, the lipases of the present invention have utility for the interesteriflcation ofoils and fats, esterification of fatty acids, digestive aids in animal feeds andin flavor enhancement (as an additive in dairy products, such as miL~) in the dairy industry.
The novel lipases, clltin~ces and enzyme compositions of the present invention are particularly useful for the removal of fatty acids from fabrics.
In this regard, they may be used in combination with dt;L~lg~ in enzymatic d~el~ellt compositions and as an additive to washing and clP~ning products. If desired, they may also be used in combination with one another and/or with other proteins, in particular enzymes including but not limited to amylases, ~lk~linP proteases and oxidases.
DeLe1~ellL compositions of the invention comprise one or more surfactants, at least one lipolytic enzyme of the invention, one or more other enzymes, de~ t builders, bleaching system, d~lelgelll additives, enzymes stabilizers. Examples of surf~rt~ntc are anionic, nonionic, cationic, or zwitterionic surf~ct~ntc, usually anionic, such as linear aL~ylbenzenesulfonate; nonionic, such as alcohol ethoxylate. Other enzymes may include amylase, protease, cellulase, peroxidase or oxidase. Suitable detel~;t;lll builders include zeolites, di- or triphosphates. Suitable delel~enLadditives include carbohydrate binders, such as dextrins or cellulose derivatives, for instance hydroxypropyl cellulose, methyl cellulose. Suitable enzymes stabilizers include propylene glycol, sorbitol or other agents known as stabilizers for enzymes. The d~lelge"l compositions have a pH between 7 and 11 in aqueous solution at the use concentration.
The dt;~elgellt composition can be either liquid or solid. It may contain the ~r~aldtion as a granulate. It may be liquid and contains the preparation as an anhydrous or subst~nti~lly anhydrous slurry.
Factors such as pH range, tolerance of emulsifiers and snrf~ct~ntc, temperature tolerance and storage capability are important considerations in the selection and development of commercially useful lipases and cutinases.
The present invention further concerns a method for removing fat stains from fabrics: this method comprises washing said fabrics with a d~;~erge, composition inclll(ling a d~;lelgellL active composition and an effective amount of the lipolytic enzyme and/or lipase and/or cutinase and/or enzyme composition of the present invention. In the washing process, the de~elgen composition used is the detergent according to the invention and in which the pH of the deLelgelll composition is between 7 and 11 and the temperature is below 60 ~C. In a plefelr~d embodiment of the invention, the washing solution contains the detergent composition according to the invention in an amount of between 1 and 5 g/l of washing solution.
Having thus described the Fusarium solanii var. minus T.92.637/1, methods for the cultivation of a biologically pure culture thereof, methods to produce and obtain the lipolytic enzymes, lipases, cutinases and enzyme compositions of the present invention thererl~ , d~ler~ellt compositions including such enzymes and/or enzyme compositions and methods for the use of the deler~e"t compositions of the present invention having such lipases, cutin~es and enzyme compositions for the removal of fatty acids from fabrics, the following examples are now presented for purposes of tr~tion only and are neither meant to be, nor should they be read as being, restrictive thereof.
Example 1 Isolation and Selection of Fusarium solanii var. minus T.92.637/1 Fusaliulll solanii var. minus T.92.637/1 was isolated from a sample of soil on an agar nutrient medium.
The agar nutrient meAium, called medium K, on which the isolates were isolated and selected was comprised of (per liter of distilled water):
5.0 grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaNO3; 1.0 gram of MgSO4,7H2O; 1.0 gram of K2HPO4; 0.5 grams of KCl; 10.0 grams of olive oil (in emulsion); 1.0 gram of polyvinylic alcohol (25/140) (in emulsion); 20 grams of agar; and 1.0 ml of 1 % (w/v) Rhodamine solution (w = weight, v = volume).
The medium K was prepared as follows:
Firstly, an olive oil emulsion was prepared. 50 ml of the distilled water was heated to 80 ~C. Then, the 1.0 gram of the polyvinylic alcohol was added to the heated distilled water in small incremental steps. Next, 10 grams of olive oil was added to the distilled water/polyvinylic alcohol and the mixture emulsified by agitation for 5 minutes at 13500 RPM
(revolutions per minute) in a blender (IJLTRA T[~RAX, mixing shaft 18 GM). An olive oil emulsion was obtained. The olive oil emulsion was then sterilized by autoclave at 121 ~C for 30 minlltes.
Secondly, an agar medium was prepared. The Bacto-Tryptone, NaNO3, MgSO4,7H20, KCl and agar were added in 950 ml of the distilled water.
The mixture was then sterili~ed by autoclave for 30 minutes at 121 ~C.
Thirdly, the Rhodamine solution was sterilized extemporaneously by filtration on 0.45 micron membranes.
Then, the olive oil emulsion and the agar were permitted to cool down to 60 ~C before the emulsion was added to the agar. The 1 ml of Rhodamine was then added thereto. The mixture was then further mixed for five minutes in a blender (IJltra Turrax equipped with 25 F mixing shaft) at 13500 RPM. Then, and before solidification, the 25 ml of the medium K so obtained was placed in respective suitable sterile Petri dishes 5 (9 cm in diameter) for subsequent use.
The samples to be screened were cultured at 25 ~C for 48 hours on the medium K and a~r~liate isolates were selected for their ability to degrade olive oil and, in the presence of Rhodamine, produce a fluorescent halo under W light.
The isolate of the present invention has been identified by its bioch~mic~l characteristics on the nutritive medium POTATO DEXTROSE
AGAR (DIFCO).
Species of Fusarium are characterized by the shape of their conidia.
Fusarium sol~nii is, above all, characterized by its formation of microconidia (phialoconidia) from very long slender conodiogenesis cell (phialide). Furthermore, those microconidia are agglomerated at the top of the conidiogenesis cell in a mucilaginous drop. Macroconidia are also typical as well as the formation of smooth to rough chlamydospore.
The ~ii.stinction between Flls~rillm solanii var. minus and other varieties of that species is based on the state of the macroconidia which are smaller in the variety minus than in other varieties.
The characteristics of the strain Fus~nllm solanii var. minus T.92.637/1 are as follows:
- colonies are fast growing, up to 80 mm in 10 days.
- aerial mycelium white to cream delicate.
- microconidia develop abundantly yet after some days of growing.
- microconidia are more or less oval to ellipsoid, thin to slightly thick walled, 8 to 13 ~m x 2.5 to 3.5 ~bm unicellular to bicellular.
- microconidia are produced from very long slender phialide (monophialidic) with a rather distinct collar, solitary or verticillate, up to 100 ~m long.
- macroconidia 1 to 3 septate, subcylindric or slightly curved, up to 30 ~m long, produced from shorter phialide, up to 20 ~m.
- chlamydospores more or less abundant, terminal or intercalary, smooth to rough-walled, single or in pairs, globose to subglobose, 5.5 ,Ibm x 8 ,um.
Fusarium solanii var. minus isolate T.92.637/1 was deposited under the Wo 96/18729 PCT/EP95/04799 provisions of the Treaty of Budapest on 23 November 1993 in the Belgian Coordinated Collections of Microorg~nisms (BCCM culture collection, MUCL) under Accession Number MUCL 38667.
Example 2 5 Obtaining Pure Culture of Fusarium solanii var. minus isolate T.92.637/1 First, a Potato Dextrose Agar (PDA) culture medium was pr~d using 39 grams/liter of Bacto Potato Dextrose Agar (DIFCO) supplemented with 12 mg of tetracycline per liter of agar culture medium.
10 ml of the medium was poured into respective 160/16 tubes and the 10 tubes were then inclinPcl and left to solidify. After soli~ifi~tinn, the various tubes were inoculated with the selected isolates which were obtained and identified as described in example 1. The culture was then developed after an incubation of 48 to 72 hours at 25 ~C. The tubes were then stored at approximately 4 ~C until use thereof.
The cultures obtained as described above were then identified as being pure cultures of Fusarium solanii var. minus isolate T.92.637/1. This identification was confirmed by the use of common taxonomic tests.
Example 3 Production of Enzymes Derived From Fusarium solanii var. minus 20 T.92.637/1 A medium was prepared comprised of (per liter of (listillPd water):
5 grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaN03; 1.0 gram of MgS04,7H20; 1.0 gram of K2HP04; 0.5 grams of KCI; 10.0 grams of soya oil; and 5.0 grams of Mazuol (Mazes Chemicals).
The medium was prepared by mixing together all of the components mentioned above, with the exception of the soya oil, and then adjusting the pH to 6.5 with 0.1 N HCI. This mixture was then sterilized by autoclave at 121 ~C for 30 minutes.
The soya oil component of the me~ m was ~ d extemporaneously 30 by the sterilization thereof by autoclave for 30 mimltes at 121 ~C.
After sterilization, the soya oil emulsion and the nutrient medium were permitted to cool down to 60 ~C before being mixed together. The resulting medium was clesign~tecl CH2 medium.
A physiological saline serum of distilled water and 0.9 % (w/v) NaCl 35 was pl~al~d and sterilized by autoclaving (at about 1.4-1.5 bars) at 121 ~C
for 30 minutes. 10 ml of this physiological serum was added to respective - inclined tubes (slants) co"l;-i"il-g the pure Fusarium culture obtained as described in Example 2. These tubes were then lightly m~nn~lly ~git~tecl to J place the Fusarium in the respective inclined tubes in suspension. These tubes con~tihlted the inoculum which were then used for subsequent inoculations.
Respective 50 ml sarnples of CH2 meAillm were then placed in respective sterile 500 ml Erlenmeyer flasks. 0.5 ml of physiological serum cont~ining the Fusarium suspension were then introduced into each tube and incubated at 25 ~C for 24 hours under agitation (orbital movement of 65 revolutions per minute with approximately 2.54 cm amplitude).
Control of the purity of the conidia was done by microscopic observation to assure noncont~min~ti-)n of the suspension of conidia.
Incubation of culture flasks, as described above, resulted in, inter alia, the extracellular production of both lipases and c--tin~es. These Fusarium lS solanii var. minus T.92.637/1 lipases and cutinases are secreted into the culture broth in the culture flask.
The presence of the lipases and c--tin~es which are naturaUy produced by Fusarium solanii var. minus T.92.637/1 in the culture broth was then confirmed, as is described below in Example 4.
Example 4 Titrimetric Lipases and Cutinases Activity Test An Erlenmeyer flask of Example 3 cont~ining a F~-s~ril-m culture was obtained and the culture broth thereof was tested for lipase activity and c~ltin~ce activity.
Determination of the lipase and cutin~e activity in the culture broth was performed as follows:
A substrate emulsion was prepared comprised of: 10 grams of Triolein (ROTH 5423); 10 grams of Gummi arabicum (Fluka No. 51 200) and 100 ml of deionized water. This mixture was em~ ified for lS Illinules in ~,~ 30 a blender (Ultra Turrax equipped with 25 F mixing shaft at 13500 rpm) in ice while being steadily stirred in a pure nitrogen gas atmosphere.
A buffer was ~urepal~d comprised of TRIS SmM, NaCl 40mM and 20 mM of CaCl2,2H2O.
The culture broth of the samples obtained as described above in Example 3 were then used to prepare active samples and inactive (for providing a standard) samples thel~rrolll. Active samples were prepared by tin~ the culture broth obtained from the samples of Example 3 by mixing approximately 1 part broth with approximately 50 parts (v/v) of the buffer (so that an enzyme concentration of approximately 2 LU/ml is provided). Inactive samples were prepared in the same manner as the active samples except that after the dilution, the inactive samples were inactivated by being heated at 110 ~C for 30 minlltes.
First, a reaction vessel (a cylindrical reaction vessel having a dimension of 24 mm x 100 mm and equipped with a pH electrode - Schott No. 6880) was filled with 10 ml of the substrate emulsion and 20 ml of the buffer.
Nitrogen gas was then pumped into the vessel to provide a pure nitrogen gas atmosphere in the vessel and the mixture stirred. The temperature of the vessel was then raised to 30 ~C.
Next, the pH of the solution was adjusted to 9.5 using 0.5N NaOH.
Then, 0.25 to 0.5 ml of the sample to be tested was added to the reaction vessel and monitoring of the titrations started. The pH of the solution was m~int~in~d at 9.5 with the addition of 0.01N NaOH, as needed. Titrations were then carried out at 30 ~C using 0.01N NaOH as the aL~ali.
After two minlltes, the total amount of added 0.01N NaOH was monitored for the next two minutes (until the titration was completed).
As used herein, LU stands for Lipase and Cutinase Units. As used herein, one lipase and cutinase unit is the amount of lipase/cutin~.~e which is nçcesc~ry to liberate one ~4mol fatty acid per minute by 1 ml of the sample being tested under the conditions of the test.
The above Titration demonstrated that the culture broth of the Fusarium isorate o~ the present invention had a lipase activity and a cutinase activity, as weU as properties of long chain hydrolysis of lipids.
Example 5 Fatty acids selectivity of lipase and cutinase Two media were prepared to identify lipase activity and cutin~ce activity.
A first medium, called medium T, was comprised of:
(per liter of distilled water) 2.5 grams of NaHCO3; 7.5 grams of Na2CO3;
10.0 grams of olive oil (in emulsion); 1.0 gram of polyvinylic alcohol (25/140) (in emulsion); 20 grams of agar; and 1.0 ml of 1 % (w/v) Rhodamine solution.
A second medium, called medium V, was comprised of:
(per liter of ~ till~d water): 2.5 grams of NaHCO3; 7.5 grams of Na2CO3; 10.0 grams of tributyrin (in emulsion); 1.0 grarn of polyvinylic alcohol (25/140) (in emulsion); 20 grams of agar.
Me lil-m V and medium T were both prepared in the same manner 5 following the protocol of and under the conditions specified in the method of preparation of the medium K, as described in example 1. Respective Petri dishes having medium V or medium T were then pr~;d.
In each of the Petri dishes of medium V and of me linm T so obtained, four separated holes were made.
S microliters of the enzyme fraction to identify were deposited in the the four holes of a Petri dish co~ g the medium T and also in the four holes of a Petri dish cont~ining the medium V.
The Petri dishes so obtained were incllb~ted at 30 ~C for 6 hours.
~rge fluorescenl zones appeared on ine m~iinm T of the Petri dishes lS around the holes, indicating a predominance of a lipase activity. This is due to the fact that a lipase has a higher selectivity toward long chain triglycerides than a cntin~e.
Large clear zones appeared on the medium V of the Petri dishes around the holes, indicating a predomin~nce of a cutinase activity. It is due to the fact that a cutinase has a higher selectivity toward short chain triglycerides than a lipase.
Example ~
Preparation of concentrated lipases solution from Fusarium solanii var.
minus T92.637/ 1 The culture flasks, obtained as described above in Example 3, were used as the inoculum for a fermentation.
13 liters of fermentation medium was ~lcpal~d comprised of (per liter of distilled water) 30 grams of tryptone (Merck 7214); S0 grams of soya oil; 5 grams of methylcellulose (Culminal 1500 PFR); 1 gram of Mazuol . 30 (Mazes Chemicals); 1 gram of NaNO3; 1 gram of MgSO4,7H20; 2 gramsof K2HP04; 0.5 grams of KCl. The pH of the medium was adjusted to 6.5 with HCl O.lN. The medium was sterilized at 121 ~C for 30 minutes.
A fermentation was then condllct~ at 25 ~C for 82 hours under constant stirring of 450 RPM (rotation per minute) and aeration of 0.3 VVM (volume per volume per minute).
A sample of the resulting ferrnenter beer was tested as described above wo 96/18729 PCTIEP95104799 in Example 4, in order to determine lipase/clltin~ce activity. Following a finding of lipase/cutinase activity, the resnlting fermenter beer was conct;~ dted as follows.
First, the pH of the fermenter beer was adjusted to 8.5 with NaOH
10N. Then, 2.0 % (w/v) of OPTIFLOC FC 205 (SOLVAY) was added together with 1.0 % (w/v) of Triton X-114 (Serva) and mixed therewith for one hour with gentle agitation. This mixture was then centrifuged at 8000 RPM (Re~km~n, rotor JA-l0) for l5 minutes at 4 ~C.
The s~ell-aL~IL was then fractionated by ammonium sulphate precipitation (saturation range: 20-60 %). A fractioned precipitate was recovered.
The fractioned precipitate was dissolved in 100 ml of an a~l~liate buffer (B R1158 SmM (ICI); CaC12 25mM; Tris/HCl 20mM, pH 7.0). A
concentrated lipases solution was thus obtained.
Example 7 Purification of the lipases A concenLldted lipases solution was obtained, as described above in Example 6. First, the lipases of this solution were isolated (extracted) from the solution by hydrophobic interaction chromatography (HIC). The extracted (isolated) lipases were then puri~led by ionic exchange chromatography (IEC) and gel filtration.
The column used for the hydrophobic interaction chromatography (HIC) is a column of Hiload 16/l0 Phenyl Sepharose High Performance (PHARMACIA 17-1085-01) with the following characteristics for the first run.
40 ml of the conce.-f .i.lPrl lipases solution as obtained in Example 6 is diluted by the starting buffer, so as to obtain 250 ml of solution to be loaded on the HlC column.
The buffers used for the HIC were the following:
Starting buffer: pot~csi~lm phosphate 20 mM pH 7.0 Elution buffer: pot~ccillm phosphate 20 mM pH 7.0 + isopropanol 30 %
(vlv) .
The flow rate was adjusted to 1.5 mVmin. The gradient was 0-l00 %.
89.3 % of the lipases were eluted in the isopropanol gradient in a 60 ml fraction.
For the second run, the 60 ml lipases fraction was then diluted with 120 ml of the starting buffer and then the 180 ml diluted solution was relo~eA on the same column in the same con(lition~.
59.4 % of the lipase was eluted in the isopl~anol gradient in a 45 ml fraction.
The 45 ml fraction was then diaf~trated on an AMICON cell (76 mm cell), equipped with a YM10 membrane, against 10 parts (v/w) of the starting buffer of the IEC chromatogphy.
Purification of the lipases from the fraction was then performed by ionic exchange chromatography (IEC) with the following characteristics.
The column used for the ionic exchange chromatography (IEC) is a column of Hiload 16/10 Q-Sepharose High Performance (PHARMACIA
17-1064-01) with the following characteristics.
The 45 ml diaf~trated solution was diluted with 195 ml of the starting buffer of the IEC chromatography.
The buffers used for the IEC were the following:
staIting buffer: Piperazine 20 mM pH 9.7 Elution buffer: Piperazine 20 mM pH 9.7 + NaCl 1.0 M (0-100 %
gradient).
The flowrate was adjusted to 3.0 ml/min.
100 % of the lipases were eluted within the NaCl gradient in a 27 ml fraction.
Fractions were then concentrated on AMICON cell (76 mm cell equipped with a YM10 membrane) and diafiltrated against 10 parts (v/w) of Tris/HCl 20 mM (pH 7.0) comprising a CaC12 5 mM buffer.
A gel filtration, with the following characteristics, was then used:
The column used was a column of Superdex 75 HR 10/30 (PHARMACIA 17-1047-01) using a buffer of CAPSO
[3-(cyclohexylamino)-2-hydroxy-1-propane sul~nic acid] 25 mM (pH 9.2) with 0.2 M NaCl.
The flowrate was adjusted to 0.5 ml/min. Fractions were obtained, each of which corresponded to a separate peak, thus showing that two ~lirrclclll lipases were obtained.
Lipase identification was made according to the method described above in Example 5. This identification revealed the presence of two separate lipases in the fraction.
The ratio of the diameters of the clear zone and the fluorescent zone CA 02208223 1997-06-ll WO 96/187~9 PCT/EP95/04799 was been determined for each fraction. For each fraction, the ratio was in the range of about 0.05 and 0.1. This result confirmed the presence of lipase activity.
Example 8 Preparation of concentrated cutinases solution from Fusarium solanii var.
minus isolate T.92.637/1 A medium was prepared comprised of (per liter of distilled water): S
grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaNO3; 1.0 gram of MgSO4,7H2O; 1.0 gram of K2HPO4; 0.5 grams of KCl; 5.0 grams of purified cutin; and 5.0 grams of Mazuol (Mazes Chemicals).
Purified Cutin was obtained from Golden Delicious Apple according to the method described by Hervé C. Gérard, in Phytocht~mic~l Analysis, Vol.3, 139-144 (1992).
The medium was prepared by mixing together all of the components mentioned above and then adjusting the pH to 6.5 with 0.1 N HCl. This mixture was then sterilized by autoclave at 121 ~C for 30 min~-tes After sterilization, the nutrient medium was permitted to cool down to 60 ~C, then to 25 ~C before ionculation.
A physiological saline serum of distilled water and 0.9 % (w/v) NaCl was ~l~alcd and sterilized by autoclaving (at about 1.4-1.5 bars) at 121 ~C
for 30 minutes. 10 ml of this physiological serum was added to respective inclin~ tubes (slants) cont~ining the pure Fusarium culture obtained as described in Example 2. These tubes were then lightly m~n~l~lly ~git~te~ to place the Pusarium in the respective inclined tubes in suspension. These tubes con~titute~l the inoculum which were then used for subsequent inoculations.
Respective 50 ml samples of the culture medium were then placed in respective sterile 500 ml Erlenmeyer flasks. 0.5 ml of physiological serum co"l;.i,-i"~ the Fusarium suspension were then introduced into each tube and incubated at 25 ~C for 24 hours under agitation (orbital movement of 65 revolutions per minute with approximately 2.54 cm amplitude).
Control of the purity of the conidia was done by microscopic observation to assure noncont~min~tion of the suspension of conidia.
The culture flask, obtained as described above, was used as the inoculum for a fermentation.
1 liter of fermentation media was prepared comprised of the medium WO 96/18729 PCT/EP9~i/04799 described above.
A fermentation was then con~lct~l at 25 ~C for 64 hours under constant stirring of 1000 RPM and aeration of 0.3 VVM.
A sample of the resulting fermenter beer was tested as described above in Example 4, in order to determine lipase/cutinase activity. FoUowing a finding of lipase/cutinase activity, the resl-lting fermenter beer was then treated as follows.
The fermenter beer was centrifuged at 8000 rpm (SW 27 rotor) for 15 minutes. The biomass was then diluted by addition of the same amount of demineralised water as the removed supernatant and then treated for 1 hour with 1 % w/w of Triton X-114 (SERVA reference 37243). The ~lihlted, treated biomass was then once again centrifuged at 8000 rpm (SW 27 rotor) for 15 min~ltes.
The supernatant was fractionated by aceton (saturation range 35-60 %).
A precipitate was recovered. The precipitate was then dissolved in 100 ml of an a~~ liate buffer (Tris/HCl 20 mM pH 7, CaC12 5 mM). A
concentrated lipases/c~ltin~es solution was so obtained.
Example 9 Separation of the lipase and the cutin~e and purification of the same A concentrated cutinases solution was obtained in the manner described above in Example 8. The cutin~e~ were then purified from the concentrated cutinases solution by Hydrophobic Interaction Chromatography (~C).
The column used is a column of Hiload 16/10 Phenyl Sepharose High Performance (PHARMACIA 17-1085-01).
The chromatography was performed having the following characteristics .
15 ml of the concentrated lipases solution, obtained as described above in Example 8, was diluted by the starting buffer, so as to obtain 250 ml of a solution to be loaded on the HIC column.
The buffers used for the HIC were potassium phosphate 20 mM pH 7.0 for the starting buffer and potassium phosphate 20 mM pH 7.0 +
isopropanol 30 % (v/v) (0-100 % gradient) for the elution buffer. The flowrate is adjusted to 1.5 ml/min.
54.6 % of the lipolytic enzymes(s) were not adsorbed on the column (240 ml active non-adsorbed fraction) while 17.2 % of the lipolytic enzymes were eluted into the isopropanol gradient (27 ml active adsorbed fraction).
CA 02208223 1997-06-ll WO 96/18729 PCT/l~P95/04799 Cutinase and lipase identifications were made according to the method described above in example 5.
A cutinase was identified to be present in the active non-adsorbed fraction and a lipase was i~lPntified to be present in the active adsorbed 5 fraction.
The ratio of the diameters of the clear zone and the fluorescent zone was then determined for each fraction. For the active non-adsorbed fraction, the ratio was in the range of about 10 and 20. This result co,~ ed the presence of clltin~e activity. For the active adsorbed fraction, the ratio was in the range of about 0.05 and 0.1. This result confirmed the presence of lipase activity.
Example 10 Molecular weight determination by SDS-PAGE analysis Purified fractions of the lipases and the cutinases of the present invention were obtained as described above in, respectively, Examples 7 and 9. These fractions were then used in an SDS-PAGE analysis for an estim~tion of the molecular weights thereof.
The estim~tion of the molecular weights of the purified enzyme samples of the present invention by the use of SDS-PAGE analysis was effectuated in denaturing conditions on polyacrylamide gel using PHARMACIA
PHASTGEL 10-15 % (w/v) by following the method described in separate technique file n~ 110 PHASTSYSTEM.
PHARMACIA LMW markers (cat n~ 17-0446-01) were used for establishing the relation of molecular weight to migration fli~t~nce. The followillg Pha~na~ia LL~1W' markers were used as moiecuiar weight standards: phosphorylase b (canine muscle) 94 kDa; albumin (bovine serum) 67 kDa; ovalbumin (chicken egg white) 43 kDa; carboanhydrase (bovine erythrocytes) 30 kDa; trypsin inhibitor (soy bean) 2Q. 1 kDa; and alpha-lactalbumin (cow milk) 14.4 kDa.
One vial, diluted in 1.5 ml of the following buffer, was also used for the sarnples: 10 mM TRIS (pH of 8.0); lmM EDTA; 2.5 % (w/v) SDS
(sodium dodecyl sulfate); 5 % (v/v) beta-mercaptoethanol and 0.01 % (w/v) bromophenol blue.
Each of the samples was first precipitated with trichloroacetic acid (final concentration 10 % w/v), and was then diluted to a concentrated of approximately 100 ,ug protein/ml, measured by the method described by CA 02208223 l997-06-ll Lowry, 1951, J.Biol. Chem., 193, pages 256-275, in the same buffer as was described above for use in rlilllting the markers. The diluted samples were then denatured by heating at 98 ~C for 15 minutes and 4 ~1 of the rlillltçd7 denatured samples were deposited on the gels. The gels were run for 60 Vh with a maximum voltage of 250 volts, a maximum power of 3 watts and maximum intensity of 10 mA.
After separation of the polypeptides, the gels were stained with Fast Coomassie as described in the Development Technique File No. 200 PHASTSYSTEM
The results of the SDS-PAGE analysis revealed four bands of about 30, 30, 22 and 60 kilodaltons (kDa).
The apparent molecular weight was found to be approximately:
- 30 kD for each of the two lipases fractions coming from Example 8, - 22 kD for the active non-adsorbed fraction co~ g the cutinase coming from Example 9, and - 60 kD for the active adsorbed fraction cont~ining the lipase coming from Example 9.
Example 1 1 Determination of the isoelectric point ~pI) bv Isoelectric focusin~
Purifled solutions of the lipases and cutinase of the present invention were obtained as described above in Examples 7 and 9. These samples were then used for determination of the isoelectric point of the lipases and cutinases of the present invention by isoelectric focusing.
Isoelectric focusing was performed using PHARMACIA
PHASTSYSTEM following the Separation Technique File No. 100 (PHASTSYSTEM ).
For this specific application, PHASTGEL IEF 3-9 was used. Pharmacia IEF markers were used (cat n~ 17-0471-01).
The gels were stained with Fast Coomassie (Development Technique File N~ 200 PHASTSYST13M ).
The isoelectric points were found to be - 6.9 for the two lipases fractions coming from Example 8, - 7.2 for the active non-adsorbed fraction cont~ining the cutinase coming from Example 9, and - 5.2 for the active adsorbed fraction cont~ining the lipase coming from Example 9.
Obviously many modifications may be made without departing from the basic spirit of the invention. Accordingly, it will be appreciated by those skilled in the art that within the scope of the appended claims, the invention may be pr~t~ti~e(l other than has been specifically described herein.
CA 02208223 l997-06-ll W096/i8729 PCT~P95/04799 nNDTCAT~NSREL~TnN~ TO A DF.POSrrF.D MICROORC.AN~ M
(~ Rulel3 ,~. n,~ ;ule Ite~w ~ell~le l t ~l~e ~lit ~L~ ;Ltli~lll lere,trell lu il~ e ~npa~c 5 .l~c 13 to 19 n. TJ)~,NTI~ A~I'ION O~' ~EPOSilT Funhcr~e~sit~ ~re i-lerlli fieLluu~l~ ~sl1ce N~UnL~ ur ~
BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM
A~dr~ss l:~r~le~ y i--~linni---- (inUILOlin,~pUstUlLLltlL~UnLlLUUn/~y) Mycothèque de l'Université Catholique de Louvain (MUCL) Place Croix du Sud, 3 BELGIUM
l~nte cldc~il AeLI~ iUIINUInlXr ~:. ADDITIONAL INDI~:ATI~>N~ /ItL~I~ hlanl ;f natappli~L7hlL~) This in~om~:~lion is t,~mlinUetl ~IU IU ~ ollul ShC.Ct [~
~usarium solanii var. minus T92.637/1 The availability of the micro-organism shall be effected only by the issue of a sample to an expert nominated by the requester.
D. DF..Sl~NATF,D~iTATF..SFORWHIC:HINDlCATlON~ R~:;M~DF,~iJllui ~ . U~L'at~tJorall~sl~na~l.5~a~L~) ~. ~71~:-'A KATF. FURNISHIN~. ~F INDICATION r; rlrulr llkml~ if no~ applir.ahlf J
n,c i l~ ow will t e subn1ir~ed lo rhc I ' Bure#u l~er (xp~cily Ihc ~cncral narur~ of sh~ ' - L~ Acccssion vu~ ypt~"si~ ) For rce,c:iving ~rl'ice ~ e ~n~ rL~r ~ c~.,.srional Rureuu u:ie only ~3 This shccl W#5 rcceivea wlth ~he ~ This shcrl w~s rccciYL~l l-y ll~e Illlelll;~ l Bure~u on:
i~. de ~ong-de ~oster J~ tl ~'i7ni olliccr !~ Au~hon edorficer Fonn r~,-r/RO/134 (JIIJY 19~
BELGIAN COORDINATED COLLECTIONS OF MICROORGANlSMS - BCCM
MUCL-COLLECTION
Page 1 of Forrn BCCM/MUCUBP/4/MUCL 38667 Receipt in the case of an original deposit Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure Receipt in the case of an original deposit issued pursuant to Rule 7.1 by the International Depositary Authority BCCMIMUCL identified at the bottom of next page International Form BCCM/MUCLIBP141MUCL 38667 To: Name of the depositor : SOLVAY (Société Anonyme) Address : 33, rue Du Prince Albert 1 050 Bruxelles 1. Identification of the microorganism:
1.1 Ide"liricdlion reference given by the cleposilvr:
T 92.637/1 1.2 Accession number given by the International Depositary Authority:
BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM
MUCL-COLLECTION
Page 2 of Form BCCM/MUCUBP/4/MUCL 38667 Receipt in the case of an original deposit Il. Scientific description and/or proposed taxonomic designation The microorganism identified under I above was acco",pan.ei by:
(mark with a cross the al, !i ' !e box(es)):
a scientific desc,i~JIion ~3 a proposed taxonomic designation lll. Receipt and acceptance This International Depositary Authority accepts the microorganism identified under I above, which was received by it on (date of original deposit) 23 november 1993 IV. International Depositary Authority Belgian Coordinated Collections of Microorganisms (BCCM) Mycothèque de l'Université Catholique de Louvain (MUCL) Place Croix du Sud 3 B-1 348 Louvain-la-Neuve Belgium Signature(s) of person(s) having the power to represent the International Depositary Authority or of authorized official(s):
Plufessor G.L. Hennebert r '-2,4- ~
Date : 8 december 1993 .,
Lipolytic enzymes, including lipases and cutin~ces, are commonly employed in dele.~ent cleaning compositions for the removal of fatty acid-based dirt and stains. As such, they have to be stable in presence of 10 these d~telgellLs~ and in particular in ~lk~line detergent solutions. Further, lipolytic enzymes are often used in combination with other enzymes, such as ~lk~lin~ proteases, amylases, oxidases and/or other proteins, thereby requiring them to also be stable in presence of these enzymes.
Lipolytic enzymes n~tllr~lly produced by various fungi are known.
Eu~ean Patent Application 0 130 064 describes an enzymatic d~;~e.gent additive including a lipase n~tllr~lly produced by the fungal strain Fusarium oxysporum.
Tnt~rn~tional Patent Application WO 90/09446 discloses an enzymatic dele.gt;ll~ composition which incllldes a cutin~e naturally produced by 20 Fusarium solanii var. pisi.
To the best of our knowledge, we are not aware of any other lipolytic enzyme naturally produced by any other strains of Fusarium. Further, we are not aware of either any lipase or cutinase which has been n~tllr~lly produced by any other species of Fusarium, including any isolate of the 25 species Fusarium solanii var. minus.
It is a prirnary object of the present invention is to provide novel lipolytic enzymes naturally produced by a fungus of the species Fusarium solanii. By the term "lipolytic enzyme" (E.C. 3.1.1), what is meant herein is a lipase (E.C. 3.1.1.3) or a cutinase (E.C. 3.1.1.50) which is capable of 30 removing stains of a fatty nature. Lipase has higher selectivity toward long chain triglycerides contained in fat than cutinase. Cutinase has higher selectivity toward short chain triglycerides contained in fat than lipase.
It is a primary object of the present invention to provide novel lipases which, when incorporated in a deLergelll composition, are capable of removing fatty acid based dirt and stains from fabrics.
It is another primary object of the present invention to provide a novel cutinase which, when incorporated in a delel~enL composition, is capable of removing fatty acid based dirt and stains from fabrics.
Sti another primary object of the present invention is to provide novel enzyme compositions of lipases and/or clltin~ces which, when incorporated in a detergent composition, are capable of removing fatty acid based dirt and stains from fabrics.
A further primary object of the present invention is to identify, isolate and provide a biologically pure culture of a novel fungus of the genus Fusarium which is capable of naturally producing the lipolytic enzymes, including the lipases and cutinases, of the present invention which lipolytic enzymes may be incorporated into the enzymatic compositions and/or the d~ler~enl compositions of the present invention.
A yet further primary object of the present invention is to provide a method for removing fatty acid-based dirt and stains from fabrics with the use of the enzymes and/or the enzyme compositions of the present mventlon.
A still yet further object of the present invention is to provide an enzymatic d~;~elgent composition capable of removing fatty acid-based dirt and stains from fabrics, which dele.~enl composition includes a lipolytic enzyme and/or a lipase and/or cutin~e and/or enzyme composition.
In accordance with the te~hing~ of the present invention, disclosed herein is a biologically pure culture of the novel isolate F~ rillm solanii var. minus T.92.637/1 and ~ c and derivatives thereof. This isolate is capable of producing the lipolytic enzymes, the lipases, the clltin~es and the enzyme compositions of the present invention.
In another aspect of the present invention, disclosed herein are novel lipolytic enzymes which are naturally produced by an isolate of the species Fusarium and, more particularly, of the species Fusarium solanii. The ~cr~illed enzymes are those lipolytic enzymes which are naturaUy produced by the isolate Fusarium solanii var. minus. The most p-cre--~d enzymes are those lipolytic enzymes which are naturally produced by the isolate Fusarium solanii var. minus T.92.637/1.
In one pl~rerled embodiment, the lipolytic enzymes of the present invention include a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa. In another pl~f~lled S embodiment, the lipolytic enzymes include a lipase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 lcDa.
In still another preferred embodiment, the lipolytic enzymes include a cntin~e having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa.
In a yet further aspect of the present invention, disclosed herein are novel enzyme compositions having at least one lipolytic enzyme which is n~ lly produced by a fungus of the species Fusarium and, more particularly, from the species Fusarium solanii. Most plerell~;d are those enzyme compositions having at least one lipolytic enzyme which is n~t~ lly produced by the isolate Fusarium solaniu var. minus T.92.637/1.
In a ~lc;r~lled embodiment, the lipolytic enzyme of the enzyme compositions is a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa. Alternatively, the lipolytic enzyme of the enzyme compositions is a lipase having an isoelectric point of 20 about 5.2 and/or an a~al~ molecular weight of about 60 IcDa. Still further alLellld~ ely, the lipolytic enzyme of the enzyme compositions is a c~-tin~e having an isoelectric point of about 7.2 and/or an a~are;lll molecular weight of about 22 kDa.
In a further ~lcfell~d embodiment, the enzyme compositions contain at 25 least two of the aforesaid lipolytic enzymes. In one ~ relled embodiment, the lipolytic enzymes are a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a lipase having an isoelectric point of about 5.2 and/or an a~par~nl molecular weight of about 60 kDa. In an alternative plerelr~d embodiment, the lipolytic enzymes are 30 a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a cutinase a having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa.
In still another alternative pl~rell~d embodiment, the lipolytic enzymes are a lipase having an isoelectric point of about 5.2 and/or an a~l~altl~t 35 molecular weight of about 60 kDa and a cutinase having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa.
Most pl~re..ed are those enzyme compositions that contain all three of the lipolytic enzymes Such compositions have a lipase having an isoelectric point of about 6 9 and/or an apparent molecular weight of about 30 kDa and a lipase having an isoelectric point of about 5 2 and/or an S apparent molecular weight of about 60 kDa and a c~ tin~e having an isoelectric point of about 7 2 and/or an apparent molecular weight of about 22 kDa In a still yet further aspect of the present invention, disclosed herein are novel enzymatic d~le.~t;--~ compositions having at least one lipolytic enzyme 10 which is n~hlr~lly produced by a fungus of the species Fusarium and, more particularly, from the species Fusarium solanii Most ~ fe-led are those enzymatic deler~en~ compositions having at least one lipolytic enzyme which is naturally produced by the isolate Fusarium solanii var minus T 92.637/1 In a ~.ere..~d embodiment, the lipolytic enzyme of the enzymatic 15 delerge..l compositions is a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa Alternatively, the lipolytic enzyme of the enzymatic d~ compositions is a lipase having an isoelectric point of about 5 2 and/or an apparent molecular weight of about 60 kDa Still further alternatively, Ihe lipolytic enzyme of the 20 enzymatic d~e-~--L compositions is a clltin~e having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa In a further ~lc;relled embodiment, the enzymatic d~
compositions contain at least two of the aforesaid lipolytic enzymes. In one p-er~ d embodiment, the lipolytic enzymes are a lipase having an 25 isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a lipase having an isoelectric point of about 5 2 and/or an a~pdl~ell~ molecular weight of about 60 kDa In an alternative ~ ;;rellcd embodiment, the lipolytic enzymes are a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a 30 clltin~e a having an isoelectric point of about 7 2 and/or an apparent molecular weight of about 22 kDa. In still another alternative ~.~rell~d embodiment, the lipolytic enzymes are a lipase having an isoelectric point of about 5 2 and/or an apparent molecular weight of about 60 kDa and a cutinase a having an isoelectric point of about 7 2 and/or an apparent 35 molecular weight of about 22 kDa Most p-efe--~d are those enzymatic detergent compositions that contain all three of the lipolytic enzymes. Such compositions have a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a lipase having an isoelectric point of about 5.2 and/or an a~e.lL molecular weight of about 60 kDa and a cutinase having an S isoelectric point of about 7.2 and/or an a~en~ molecular weight of about 22 kDa.
The enzymatic detergent compositions of the present invention are all capable of removing fatty acid-based dirt and stains from fabrics.
These and further objects and advantages of the present invention will become readily apparent from a reading of the following description.
The novel isolate of the present invention is a biologically pure culture of the isolate Fusarium solanii var. minus T.92.637/1.
The fungus Fusarium solanii var. minus T.92.637/1 was deposited under the provisions of the Budapest Treaty on 23 November 1993 with the BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS
(MUCL: THE AGRICULTURAL AND INDUSTRIAL FILAMENTOUS
AND YEAST FUNGI COLLECTION, Mycothéque de l'Université
Catholique de Louvain, Place Croix du Sud 3, B-1348 Louvain-La-Neuve, Belgium under accession number MUCL 38667).
The morphological characteristics of Fusarium solanii var. minus T.92.637/1 are set forth in detail below in Example 1.
Natural and artificial mllt~ntc and derivatives of Fusarium solanii var.
minus T.92.637/1 may be obtained by natural modifications or by genetic morlifi~tions thereof. Such well known methods as, for example, by X-ray techniques, ultraviolet irradiation, chemical mutagens or genetic engineering can all be employed to obtain such mllt~ntC and derivatives. It is to be expressly understood that such mutants and derivatives are also contemplated and encomp~csed by the present invention.
The novel lipolytic enzymes, lipases, cutinases and enzyme compositions of the present invention are n~t~ lly produced by a fungus of the genus Fusarium and, more particularly, from the isolate Fusarium solanii var. minus T.92.637/1.
The lipolytic enzymes, lipases, c~ltin~ces and enzyme compositions of the present invention are naturally produced during the growth of the fungus and are subst~nti~lly extracellularly excreted (secreted) into the medium.
These lipolytic enzymes, lipases, cutinases and enzyme compositions may be recovered using normal techni(lues, generally comprising centrifugation to remove the fungal cells, followed by concentration by ultraf~tration. The crude lipolytic enzymes, lipases, cutin~ces and enzyme compositions obtained in this way can then be dried. An additional purific~tion stage is also possible in order to separate cont~min~tin~ proteins from the lipolytic enzymes, lipases, cutin~ces and enzyme compositions of the present nvention.
One of the lipases of the present invention is characterized by having an isoelectric point of about 5.2 and an apparent molecular weight of about 60 kDa. Another of the lipases of the present invention is characterized by having an isoelectric point of about 6.9 and an apparent molecular weight of about 30 kDa.
The cutinase of the present invention is characterized by having an isoelectric point of about 7.2 and an appalel.~ molecular weight of about 22kD.
The enzyme compositions of the present invention are comprised of the lipolytic enzymes and/or lipases and/or cutin~ces of the present invention.
More particularly, the enzyme compositions include either at least one lipase and/or at least one cutin~ce naturally produced by Fusarium solanii var. minus. T.92.637/1. In a pl~relled embodiment, the enzyme composition of the present invention includes at least one of the lipases and at least one of the cutinases naturally produced by Fusarium solanii var.
minus. T.92.637/1. In another embo-liment, the enzyme composition incllldes the lipases which are naturally produced by Fusarium solanii var.
minus. T.92.637/1. Most ~l~;Çt;lled, is that enzyme composition includes the lipases and the cutin~ces naturally produced by Fusarium solanii var.
minus. T.92.637/1.
The lipolytic enzymes (lipases and cutinases) and enzyme compositions of the present invention have use in a number of varying applications for hydrolyzing and modifying fats, ranging from, inter alia, oleochemistry processing applications to soap and dt;ler~en~ compositions. For example, the lipases of the present invention have utility for the interesteriflcation ofoils and fats, esterification of fatty acids, digestive aids in animal feeds andin flavor enhancement (as an additive in dairy products, such as miL~) in the dairy industry.
The novel lipases, clltin~ces and enzyme compositions of the present invention are particularly useful for the removal of fatty acids from fabrics.
In this regard, they may be used in combination with dt;L~lg~ in enzymatic d~el~ellt compositions and as an additive to washing and clP~ning products. If desired, they may also be used in combination with one another and/or with other proteins, in particular enzymes including but not limited to amylases, ~lk~linP proteases and oxidases.
DeLe1~ellL compositions of the invention comprise one or more surfactants, at least one lipolytic enzyme of the invention, one or more other enzymes, de~ t builders, bleaching system, d~lelgelll additives, enzymes stabilizers. Examples of surf~rt~ntc are anionic, nonionic, cationic, or zwitterionic surf~ct~ntc, usually anionic, such as linear aL~ylbenzenesulfonate; nonionic, such as alcohol ethoxylate. Other enzymes may include amylase, protease, cellulase, peroxidase or oxidase. Suitable detel~;t;lll builders include zeolites, di- or triphosphates. Suitable delel~enLadditives include carbohydrate binders, such as dextrins or cellulose derivatives, for instance hydroxypropyl cellulose, methyl cellulose. Suitable enzymes stabilizers include propylene glycol, sorbitol or other agents known as stabilizers for enzymes. The d~lelge"l compositions have a pH between 7 and 11 in aqueous solution at the use concentration.
The dt;~elgellt composition can be either liquid or solid. It may contain the ~r~aldtion as a granulate. It may be liquid and contains the preparation as an anhydrous or subst~nti~lly anhydrous slurry.
Factors such as pH range, tolerance of emulsifiers and snrf~ct~ntc, temperature tolerance and storage capability are important considerations in the selection and development of commercially useful lipases and cutinases.
The present invention further concerns a method for removing fat stains from fabrics: this method comprises washing said fabrics with a d~;~erge, composition inclll(ling a d~;lelgellL active composition and an effective amount of the lipolytic enzyme and/or lipase and/or cutinase and/or enzyme composition of the present invention. In the washing process, the de~elgen composition used is the detergent according to the invention and in which the pH of the deLelgelll composition is between 7 and 11 and the temperature is below 60 ~C. In a plefelr~d embodiment of the invention, the washing solution contains the detergent composition according to the invention in an amount of between 1 and 5 g/l of washing solution.
Having thus described the Fusarium solanii var. minus T.92.637/1, methods for the cultivation of a biologically pure culture thereof, methods to produce and obtain the lipolytic enzymes, lipases, cutinases and enzyme compositions of the present invention thererl~ , d~ler~ellt compositions including such enzymes and/or enzyme compositions and methods for the use of the deler~e"t compositions of the present invention having such lipases, cutin~es and enzyme compositions for the removal of fatty acids from fabrics, the following examples are now presented for purposes of tr~tion only and are neither meant to be, nor should they be read as being, restrictive thereof.
Example 1 Isolation and Selection of Fusarium solanii var. minus T.92.637/1 Fusaliulll solanii var. minus T.92.637/1 was isolated from a sample of soil on an agar nutrient medium.
The agar nutrient meAium, called medium K, on which the isolates were isolated and selected was comprised of (per liter of distilled water):
5.0 grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaNO3; 1.0 gram of MgSO4,7H2O; 1.0 gram of K2HPO4; 0.5 grams of KCl; 10.0 grams of olive oil (in emulsion); 1.0 gram of polyvinylic alcohol (25/140) (in emulsion); 20 grams of agar; and 1.0 ml of 1 % (w/v) Rhodamine solution (w = weight, v = volume).
The medium K was prepared as follows:
Firstly, an olive oil emulsion was prepared. 50 ml of the distilled water was heated to 80 ~C. Then, the 1.0 gram of the polyvinylic alcohol was added to the heated distilled water in small incremental steps. Next, 10 grams of olive oil was added to the distilled water/polyvinylic alcohol and the mixture emulsified by agitation for 5 minutes at 13500 RPM
(revolutions per minute) in a blender (IJLTRA T[~RAX, mixing shaft 18 GM). An olive oil emulsion was obtained. The olive oil emulsion was then sterilized by autoclave at 121 ~C for 30 minlltes.
Secondly, an agar medium was prepared. The Bacto-Tryptone, NaNO3, MgSO4,7H20, KCl and agar were added in 950 ml of the distilled water.
The mixture was then sterili~ed by autoclave for 30 minutes at 121 ~C.
Thirdly, the Rhodamine solution was sterilized extemporaneously by filtration on 0.45 micron membranes.
Then, the olive oil emulsion and the agar were permitted to cool down to 60 ~C before the emulsion was added to the agar. The 1 ml of Rhodamine was then added thereto. The mixture was then further mixed for five minutes in a blender (IJltra Turrax equipped with 25 F mixing shaft) at 13500 RPM. Then, and before solidification, the 25 ml of the medium K so obtained was placed in respective suitable sterile Petri dishes 5 (9 cm in diameter) for subsequent use.
The samples to be screened were cultured at 25 ~C for 48 hours on the medium K and a~r~liate isolates were selected for their ability to degrade olive oil and, in the presence of Rhodamine, produce a fluorescent halo under W light.
The isolate of the present invention has been identified by its bioch~mic~l characteristics on the nutritive medium POTATO DEXTROSE
AGAR (DIFCO).
Species of Fusarium are characterized by the shape of their conidia.
Fusarium sol~nii is, above all, characterized by its formation of microconidia (phialoconidia) from very long slender conodiogenesis cell (phialide). Furthermore, those microconidia are agglomerated at the top of the conidiogenesis cell in a mucilaginous drop. Macroconidia are also typical as well as the formation of smooth to rough chlamydospore.
The ~ii.stinction between Flls~rillm solanii var. minus and other varieties of that species is based on the state of the macroconidia which are smaller in the variety minus than in other varieties.
The characteristics of the strain Fus~nllm solanii var. minus T.92.637/1 are as follows:
- colonies are fast growing, up to 80 mm in 10 days.
- aerial mycelium white to cream delicate.
- microconidia develop abundantly yet after some days of growing.
- microconidia are more or less oval to ellipsoid, thin to slightly thick walled, 8 to 13 ~m x 2.5 to 3.5 ~bm unicellular to bicellular.
- microconidia are produced from very long slender phialide (monophialidic) with a rather distinct collar, solitary or verticillate, up to 100 ~m long.
- macroconidia 1 to 3 septate, subcylindric or slightly curved, up to 30 ~m long, produced from shorter phialide, up to 20 ~m.
- chlamydospores more or less abundant, terminal or intercalary, smooth to rough-walled, single or in pairs, globose to subglobose, 5.5 ,Ibm x 8 ,um.
Fusarium solanii var. minus isolate T.92.637/1 was deposited under the Wo 96/18729 PCT/EP95/04799 provisions of the Treaty of Budapest on 23 November 1993 in the Belgian Coordinated Collections of Microorg~nisms (BCCM culture collection, MUCL) under Accession Number MUCL 38667.
Example 2 5 Obtaining Pure Culture of Fusarium solanii var. minus isolate T.92.637/1 First, a Potato Dextrose Agar (PDA) culture medium was pr~d using 39 grams/liter of Bacto Potato Dextrose Agar (DIFCO) supplemented with 12 mg of tetracycline per liter of agar culture medium.
10 ml of the medium was poured into respective 160/16 tubes and the 10 tubes were then inclinPcl and left to solidify. After soli~ifi~tinn, the various tubes were inoculated with the selected isolates which were obtained and identified as described in example 1. The culture was then developed after an incubation of 48 to 72 hours at 25 ~C. The tubes were then stored at approximately 4 ~C until use thereof.
The cultures obtained as described above were then identified as being pure cultures of Fusarium solanii var. minus isolate T.92.637/1. This identification was confirmed by the use of common taxonomic tests.
Example 3 Production of Enzymes Derived From Fusarium solanii var. minus 20 T.92.637/1 A medium was prepared comprised of (per liter of (listillPd water):
5 grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaN03; 1.0 gram of MgS04,7H20; 1.0 gram of K2HP04; 0.5 grams of KCI; 10.0 grams of soya oil; and 5.0 grams of Mazuol (Mazes Chemicals).
The medium was prepared by mixing together all of the components mentioned above, with the exception of the soya oil, and then adjusting the pH to 6.5 with 0.1 N HCI. This mixture was then sterilized by autoclave at 121 ~C for 30 minutes.
The soya oil component of the me~ m was ~ d extemporaneously 30 by the sterilization thereof by autoclave for 30 mimltes at 121 ~C.
After sterilization, the soya oil emulsion and the nutrient medium were permitted to cool down to 60 ~C before being mixed together. The resulting medium was clesign~tecl CH2 medium.
A physiological saline serum of distilled water and 0.9 % (w/v) NaCl 35 was pl~al~d and sterilized by autoclaving (at about 1.4-1.5 bars) at 121 ~C
for 30 minutes. 10 ml of this physiological serum was added to respective - inclined tubes (slants) co"l;-i"il-g the pure Fusarium culture obtained as described in Example 2. These tubes were then lightly m~nn~lly ~git~tecl to J place the Fusarium in the respective inclined tubes in suspension. These tubes con~tihlted the inoculum which were then used for subsequent inoculations.
Respective 50 ml sarnples of CH2 meAillm were then placed in respective sterile 500 ml Erlenmeyer flasks. 0.5 ml of physiological serum cont~ining the Fusarium suspension were then introduced into each tube and incubated at 25 ~C for 24 hours under agitation (orbital movement of 65 revolutions per minute with approximately 2.54 cm amplitude).
Control of the purity of the conidia was done by microscopic observation to assure noncont~min~ti-)n of the suspension of conidia.
Incubation of culture flasks, as described above, resulted in, inter alia, the extracellular production of both lipases and c--tin~es. These Fusarium lS solanii var. minus T.92.637/1 lipases and cutinases are secreted into the culture broth in the culture flask.
The presence of the lipases and c--tin~es which are naturaUy produced by Fusarium solanii var. minus T.92.637/1 in the culture broth was then confirmed, as is described below in Example 4.
Example 4 Titrimetric Lipases and Cutinases Activity Test An Erlenmeyer flask of Example 3 cont~ining a F~-s~ril-m culture was obtained and the culture broth thereof was tested for lipase activity and c~ltin~ce activity.
Determination of the lipase and cutin~e activity in the culture broth was performed as follows:
A substrate emulsion was prepared comprised of: 10 grams of Triolein (ROTH 5423); 10 grams of Gummi arabicum (Fluka No. 51 200) and 100 ml of deionized water. This mixture was em~ ified for lS Illinules in ~,~ 30 a blender (Ultra Turrax equipped with 25 F mixing shaft at 13500 rpm) in ice while being steadily stirred in a pure nitrogen gas atmosphere.
A buffer was ~urepal~d comprised of TRIS SmM, NaCl 40mM and 20 mM of CaCl2,2H2O.
The culture broth of the samples obtained as described above in Example 3 were then used to prepare active samples and inactive (for providing a standard) samples thel~rrolll. Active samples were prepared by tin~ the culture broth obtained from the samples of Example 3 by mixing approximately 1 part broth with approximately 50 parts (v/v) of the buffer (so that an enzyme concentration of approximately 2 LU/ml is provided). Inactive samples were prepared in the same manner as the active samples except that after the dilution, the inactive samples were inactivated by being heated at 110 ~C for 30 minlltes.
First, a reaction vessel (a cylindrical reaction vessel having a dimension of 24 mm x 100 mm and equipped with a pH electrode - Schott No. 6880) was filled with 10 ml of the substrate emulsion and 20 ml of the buffer.
Nitrogen gas was then pumped into the vessel to provide a pure nitrogen gas atmosphere in the vessel and the mixture stirred. The temperature of the vessel was then raised to 30 ~C.
Next, the pH of the solution was adjusted to 9.5 using 0.5N NaOH.
Then, 0.25 to 0.5 ml of the sample to be tested was added to the reaction vessel and monitoring of the titrations started. The pH of the solution was m~int~in~d at 9.5 with the addition of 0.01N NaOH, as needed. Titrations were then carried out at 30 ~C using 0.01N NaOH as the aL~ali.
After two minlltes, the total amount of added 0.01N NaOH was monitored for the next two minutes (until the titration was completed).
As used herein, LU stands for Lipase and Cutinase Units. As used herein, one lipase and cutinase unit is the amount of lipase/cutin~.~e which is nçcesc~ry to liberate one ~4mol fatty acid per minute by 1 ml of the sample being tested under the conditions of the test.
The above Titration demonstrated that the culture broth of the Fusarium isorate o~ the present invention had a lipase activity and a cutinase activity, as weU as properties of long chain hydrolysis of lipids.
Example 5 Fatty acids selectivity of lipase and cutinase Two media were prepared to identify lipase activity and cutin~ce activity.
A first medium, called medium T, was comprised of:
(per liter of distilled water) 2.5 grams of NaHCO3; 7.5 grams of Na2CO3;
10.0 grams of olive oil (in emulsion); 1.0 gram of polyvinylic alcohol (25/140) (in emulsion); 20 grams of agar; and 1.0 ml of 1 % (w/v) Rhodamine solution.
A second medium, called medium V, was comprised of:
(per liter of ~ till~d water): 2.5 grams of NaHCO3; 7.5 grams of Na2CO3; 10.0 grams of tributyrin (in emulsion); 1.0 grarn of polyvinylic alcohol (25/140) (in emulsion); 20 grams of agar.
Me lil-m V and medium T were both prepared in the same manner 5 following the protocol of and under the conditions specified in the method of preparation of the medium K, as described in example 1. Respective Petri dishes having medium V or medium T were then pr~;d.
In each of the Petri dishes of medium V and of me linm T so obtained, four separated holes were made.
S microliters of the enzyme fraction to identify were deposited in the the four holes of a Petri dish co~ g the medium T and also in the four holes of a Petri dish cont~ining the medium V.
The Petri dishes so obtained were incllb~ted at 30 ~C for 6 hours.
~rge fluorescenl zones appeared on ine m~iinm T of the Petri dishes lS around the holes, indicating a predominance of a lipase activity. This is due to the fact that a lipase has a higher selectivity toward long chain triglycerides than a cntin~e.
Large clear zones appeared on the medium V of the Petri dishes around the holes, indicating a predomin~nce of a cutinase activity. It is due to the fact that a cutinase has a higher selectivity toward short chain triglycerides than a lipase.
Example ~
Preparation of concentrated lipases solution from Fusarium solanii var.
minus T92.637/ 1 The culture flasks, obtained as described above in Example 3, were used as the inoculum for a fermentation.
13 liters of fermentation medium was ~lcpal~d comprised of (per liter of distilled water) 30 grams of tryptone (Merck 7214); S0 grams of soya oil; 5 grams of methylcellulose (Culminal 1500 PFR); 1 gram of Mazuol . 30 (Mazes Chemicals); 1 gram of NaNO3; 1 gram of MgSO4,7H20; 2 gramsof K2HP04; 0.5 grams of KCl. The pH of the medium was adjusted to 6.5 with HCl O.lN. The medium was sterilized at 121 ~C for 30 minutes.
A fermentation was then condllct~ at 25 ~C for 82 hours under constant stirring of 450 RPM (rotation per minute) and aeration of 0.3 VVM (volume per volume per minute).
A sample of the resulting ferrnenter beer was tested as described above wo 96/18729 PCTIEP95104799 in Example 4, in order to determine lipase/clltin~ce activity. Following a finding of lipase/cutinase activity, the resnlting fermenter beer was conct;~ dted as follows.
First, the pH of the fermenter beer was adjusted to 8.5 with NaOH
10N. Then, 2.0 % (w/v) of OPTIFLOC FC 205 (SOLVAY) was added together with 1.0 % (w/v) of Triton X-114 (Serva) and mixed therewith for one hour with gentle agitation. This mixture was then centrifuged at 8000 RPM (Re~km~n, rotor JA-l0) for l5 minutes at 4 ~C.
The s~ell-aL~IL was then fractionated by ammonium sulphate precipitation (saturation range: 20-60 %). A fractioned precipitate was recovered.
The fractioned precipitate was dissolved in 100 ml of an a~l~liate buffer (B R1158 SmM (ICI); CaC12 25mM; Tris/HCl 20mM, pH 7.0). A
concentrated lipases solution was thus obtained.
Example 7 Purification of the lipases A concenLldted lipases solution was obtained, as described above in Example 6. First, the lipases of this solution were isolated (extracted) from the solution by hydrophobic interaction chromatography (HIC). The extracted (isolated) lipases were then puri~led by ionic exchange chromatography (IEC) and gel filtration.
The column used for the hydrophobic interaction chromatography (HIC) is a column of Hiload 16/l0 Phenyl Sepharose High Performance (PHARMACIA 17-1085-01) with the following characteristics for the first run.
40 ml of the conce.-f .i.lPrl lipases solution as obtained in Example 6 is diluted by the starting buffer, so as to obtain 250 ml of solution to be loaded on the HlC column.
The buffers used for the HIC were the following:
Starting buffer: pot~csi~lm phosphate 20 mM pH 7.0 Elution buffer: pot~ccillm phosphate 20 mM pH 7.0 + isopropanol 30 %
(vlv) .
The flow rate was adjusted to 1.5 mVmin. The gradient was 0-l00 %.
89.3 % of the lipases were eluted in the isopropanol gradient in a 60 ml fraction.
For the second run, the 60 ml lipases fraction was then diluted with 120 ml of the starting buffer and then the 180 ml diluted solution was relo~eA on the same column in the same con(lition~.
59.4 % of the lipase was eluted in the isopl~anol gradient in a 45 ml fraction.
The 45 ml fraction was then diaf~trated on an AMICON cell (76 mm cell), equipped with a YM10 membrane, against 10 parts (v/w) of the starting buffer of the IEC chromatogphy.
Purification of the lipases from the fraction was then performed by ionic exchange chromatography (IEC) with the following characteristics.
The column used for the ionic exchange chromatography (IEC) is a column of Hiload 16/10 Q-Sepharose High Performance (PHARMACIA
17-1064-01) with the following characteristics.
The 45 ml diaf~trated solution was diluted with 195 ml of the starting buffer of the IEC chromatography.
The buffers used for the IEC were the following:
staIting buffer: Piperazine 20 mM pH 9.7 Elution buffer: Piperazine 20 mM pH 9.7 + NaCl 1.0 M (0-100 %
gradient).
The flowrate was adjusted to 3.0 ml/min.
100 % of the lipases were eluted within the NaCl gradient in a 27 ml fraction.
Fractions were then concentrated on AMICON cell (76 mm cell equipped with a YM10 membrane) and diafiltrated against 10 parts (v/w) of Tris/HCl 20 mM (pH 7.0) comprising a CaC12 5 mM buffer.
A gel filtration, with the following characteristics, was then used:
The column used was a column of Superdex 75 HR 10/30 (PHARMACIA 17-1047-01) using a buffer of CAPSO
[3-(cyclohexylamino)-2-hydroxy-1-propane sul~nic acid] 25 mM (pH 9.2) with 0.2 M NaCl.
The flowrate was adjusted to 0.5 ml/min. Fractions were obtained, each of which corresponded to a separate peak, thus showing that two ~lirrclclll lipases were obtained.
Lipase identification was made according to the method described above in Example 5. This identification revealed the presence of two separate lipases in the fraction.
The ratio of the diameters of the clear zone and the fluorescent zone CA 02208223 1997-06-ll WO 96/187~9 PCT/EP95/04799 was been determined for each fraction. For each fraction, the ratio was in the range of about 0.05 and 0.1. This result confirmed the presence of lipase activity.
Example 8 Preparation of concentrated cutinases solution from Fusarium solanii var.
minus isolate T.92.637/1 A medium was prepared comprised of (per liter of distilled water): S
grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaNO3; 1.0 gram of MgSO4,7H2O; 1.0 gram of K2HPO4; 0.5 grams of KCl; 5.0 grams of purified cutin; and 5.0 grams of Mazuol (Mazes Chemicals).
Purified Cutin was obtained from Golden Delicious Apple according to the method described by Hervé C. Gérard, in Phytocht~mic~l Analysis, Vol.3, 139-144 (1992).
The medium was prepared by mixing together all of the components mentioned above and then adjusting the pH to 6.5 with 0.1 N HCl. This mixture was then sterilized by autoclave at 121 ~C for 30 min~-tes After sterilization, the nutrient medium was permitted to cool down to 60 ~C, then to 25 ~C before ionculation.
A physiological saline serum of distilled water and 0.9 % (w/v) NaCl was ~l~alcd and sterilized by autoclaving (at about 1.4-1.5 bars) at 121 ~C
for 30 minutes. 10 ml of this physiological serum was added to respective inclin~ tubes (slants) cont~ining the pure Fusarium culture obtained as described in Example 2. These tubes were then lightly m~n~l~lly ~git~te~ to place the Pusarium in the respective inclined tubes in suspension. These tubes con~titute~l the inoculum which were then used for subsequent inoculations.
Respective 50 ml samples of the culture medium were then placed in respective sterile 500 ml Erlenmeyer flasks. 0.5 ml of physiological serum co"l;.i,-i"~ the Fusarium suspension were then introduced into each tube and incubated at 25 ~C for 24 hours under agitation (orbital movement of 65 revolutions per minute with approximately 2.54 cm amplitude).
Control of the purity of the conidia was done by microscopic observation to assure noncont~min~tion of the suspension of conidia.
The culture flask, obtained as described above, was used as the inoculum for a fermentation.
1 liter of fermentation media was prepared comprised of the medium WO 96/18729 PCT/EP9~i/04799 described above.
A fermentation was then con~lct~l at 25 ~C for 64 hours under constant stirring of 1000 RPM and aeration of 0.3 VVM.
A sample of the resulting fermenter beer was tested as described above in Example 4, in order to determine lipase/cutinase activity. FoUowing a finding of lipase/cutinase activity, the resl-lting fermenter beer was then treated as follows.
The fermenter beer was centrifuged at 8000 rpm (SW 27 rotor) for 15 minutes. The biomass was then diluted by addition of the same amount of demineralised water as the removed supernatant and then treated for 1 hour with 1 % w/w of Triton X-114 (SERVA reference 37243). The ~lihlted, treated biomass was then once again centrifuged at 8000 rpm (SW 27 rotor) for 15 min~ltes.
The supernatant was fractionated by aceton (saturation range 35-60 %).
A precipitate was recovered. The precipitate was then dissolved in 100 ml of an a~~ liate buffer (Tris/HCl 20 mM pH 7, CaC12 5 mM). A
concentrated lipases/c~ltin~es solution was so obtained.
Example 9 Separation of the lipase and the cutin~e and purification of the same A concentrated cutinases solution was obtained in the manner described above in Example 8. The cutin~e~ were then purified from the concentrated cutinases solution by Hydrophobic Interaction Chromatography (~C).
The column used is a column of Hiload 16/10 Phenyl Sepharose High Performance (PHARMACIA 17-1085-01).
The chromatography was performed having the following characteristics .
15 ml of the concentrated lipases solution, obtained as described above in Example 8, was diluted by the starting buffer, so as to obtain 250 ml of a solution to be loaded on the HIC column.
The buffers used for the HIC were potassium phosphate 20 mM pH 7.0 for the starting buffer and potassium phosphate 20 mM pH 7.0 +
isopropanol 30 % (v/v) (0-100 % gradient) for the elution buffer. The flowrate is adjusted to 1.5 ml/min.
54.6 % of the lipolytic enzymes(s) were not adsorbed on the column (240 ml active non-adsorbed fraction) while 17.2 % of the lipolytic enzymes were eluted into the isopropanol gradient (27 ml active adsorbed fraction).
CA 02208223 1997-06-ll WO 96/18729 PCT/l~P95/04799 Cutinase and lipase identifications were made according to the method described above in example 5.
A cutinase was identified to be present in the active non-adsorbed fraction and a lipase was i~lPntified to be present in the active adsorbed 5 fraction.
The ratio of the diameters of the clear zone and the fluorescent zone was then determined for each fraction. For the active non-adsorbed fraction, the ratio was in the range of about 10 and 20. This result co,~ ed the presence of clltin~e activity. For the active adsorbed fraction, the ratio was in the range of about 0.05 and 0.1. This result confirmed the presence of lipase activity.
Example 10 Molecular weight determination by SDS-PAGE analysis Purified fractions of the lipases and the cutinases of the present invention were obtained as described above in, respectively, Examples 7 and 9. These fractions were then used in an SDS-PAGE analysis for an estim~tion of the molecular weights thereof.
The estim~tion of the molecular weights of the purified enzyme samples of the present invention by the use of SDS-PAGE analysis was effectuated in denaturing conditions on polyacrylamide gel using PHARMACIA
PHASTGEL 10-15 % (w/v) by following the method described in separate technique file n~ 110 PHASTSYSTEM.
PHARMACIA LMW markers (cat n~ 17-0446-01) were used for establishing the relation of molecular weight to migration fli~t~nce. The followillg Pha~na~ia LL~1W' markers were used as moiecuiar weight standards: phosphorylase b (canine muscle) 94 kDa; albumin (bovine serum) 67 kDa; ovalbumin (chicken egg white) 43 kDa; carboanhydrase (bovine erythrocytes) 30 kDa; trypsin inhibitor (soy bean) 2Q. 1 kDa; and alpha-lactalbumin (cow milk) 14.4 kDa.
One vial, diluted in 1.5 ml of the following buffer, was also used for the sarnples: 10 mM TRIS (pH of 8.0); lmM EDTA; 2.5 % (w/v) SDS
(sodium dodecyl sulfate); 5 % (v/v) beta-mercaptoethanol and 0.01 % (w/v) bromophenol blue.
Each of the samples was first precipitated with trichloroacetic acid (final concentration 10 % w/v), and was then diluted to a concentrated of approximately 100 ,ug protein/ml, measured by the method described by CA 02208223 l997-06-ll Lowry, 1951, J.Biol. Chem., 193, pages 256-275, in the same buffer as was described above for use in rlilllting the markers. The diluted samples were then denatured by heating at 98 ~C for 15 minutes and 4 ~1 of the rlillltçd7 denatured samples were deposited on the gels. The gels were run for 60 Vh with a maximum voltage of 250 volts, a maximum power of 3 watts and maximum intensity of 10 mA.
After separation of the polypeptides, the gels were stained with Fast Coomassie as described in the Development Technique File No. 200 PHASTSYSTEM
The results of the SDS-PAGE analysis revealed four bands of about 30, 30, 22 and 60 kilodaltons (kDa).
The apparent molecular weight was found to be approximately:
- 30 kD for each of the two lipases fractions coming from Example 8, - 22 kD for the active non-adsorbed fraction co~ g the cutinase coming from Example 9, and - 60 kD for the active adsorbed fraction cont~ining the lipase coming from Example 9.
Example 1 1 Determination of the isoelectric point ~pI) bv Isoelectric focusin~
Purifled solutions of the lipases and cutinase of the present invention were obtained as described above in Examples 7 and 9. These samples were then used for determination of the isoelectric point of the lipases and cutinases of the present invention by isoelectric focusing.
Isoelectric focusing was performed using PHARMACIA
PHASTSYSTEM following the Separation Technique File No. 100 (PHASTSYSTEM ).
For this specific application, PHASTGEL IEF 3-9 was used. Pharmacia IEF markers were used (cat n~ 17-0471-01).
The gels were stained with Fast Coomassie (Development Technique File N~ 200 PHASTSYST13M ).
The isoelectric points were found to be - 6.9 for the two lipases fractions coming from Example 8, - 7.2 for the active non-adsorbed fraction cont~ining the cutinase coming from Example 9, and - 5.2 for the active adsorbed fraction cont~ining the lipase coming from Example 9.
Obviously many modifications may be made without departing from the basic spirit of the invention. Accordingly, it will be appreciated by those skilled in the art that within the scope of the appended claims, the invention may be pr~t~ti~e(l other than has been specifically described herein.
CA 02208223 l997-06-ll W096/i8729 PCT~P95/04799 nNDTCAT~NSREL~TnN~ TO A DF.POSrrF.D MICROORC.AN~ M
(~ Rulel3 ,~. n,~ ;ule Ite~w ~ell~le l t ~l~e ~lit ~L~ ;Ltli~lll lere,trell lu il~ e ~npa~c 5 .l~c 13 to 19 n. TJ)~,NTI~ A~I'ION O~' ~EPOSilT Funhcr~e~sit~ ~re i-lerlli fieLluu~l~ ~sl1ce N~UnL~ ur ~
BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM
A~dr~ss l:~r~le~ y i--~linni---- (inUILOlin,~pUstUlLLltlL~UnLlLUUn/~y) Mycothèque de l'Université Catholique de Louvain (MUCL) Place Croix du Sud, 3 BELGIUM
l~nte cldc~il AeLI~ iUIINUInlXr ~:. ADDITIONAL INDI~:ATI~>N~ /ItL~I~ hlanl ;f natappli~L7hlL~) This in~om~:~lion is t,~mlinUetl ~IU IU ~ ollul ShC.Ct [~
~usarium solanii var. minus T92.637/1 The availability of the micro-organism shall be effected only by the issue of a sample to an expert nominated by the requester.
D. DF..Sl~NATF,D~iTATF..SFORWHIC:HINDlCATlON~ R~:;M~DF,~iJllui ~ . U~L'at~tJorall~sl~na~l.5~a~L~) ~. ~71~:-'A KATF. FURNISHIN~. ~F INDICATION r; rlrulr llkml~ if no~ applir.ahlf J
n,c i l~ ow will t e subn1ir~ed lo rhc I ' Bure#u l~er (xp~cily Ihc ~cncral narur~ of sh~ ' - L~ Acccssion vu~ ypt~"si~ ) For rce,c:iving ~rl'ice ~ e ~n~ rL~r ~ c~.,.srional Rureuu u:ie only ~3 This shccl W#5 rcceivea wlth ~he ~ This shcrl w~s rccciYL~l l-y ll~e Illlelll;~ l Bure~u on:
i~. de ~ong-de ~oster J~ tl ~'i7ni olliccr !~ Au~hon edorficer Fonn r~,-r/RO/134 (JIIJY 19~
BELGIAN COORDINATED COLLECTIONS OF MICROORGANlSMS - BCCM
MUCL-COLLECTION
Page 1 of Forrn BCCM/MUCUBP/4/MUCL 38667 Receipt in the case of an original deposit Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure Receipt in the case of an original deposit issued pursuant to Rule 7.1 by the International Depositary Authority BCCMIMUCL identified at the bottom of next page International Form BCCM/MUCLIBP141MUCL 38667 To: Name of the depositor : SOLVAY (Société Anonyme) Address : 33, rue Du Prince Albert 1 050 Bruxelles 1. Identification of the microorganism:
1.1 Ide"liricdlion reference given by the cleposilvr:
T 92.637/1 1.2 Accession number given by the International Depositary Authority:
BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM
MUCL-COLLECTION
Page 2 of Form BCCM/MUCUBP/4/MUCL 38667 Receipt in the case of an original deposit Il. Scientific description and/or proposed taxonomic designation The microorganism identified under I above was acco",pan.ei by:
(mark with a cross the al, !i ' !e box(es)):
a scientific desc,i~JIion ~3 a proposed taxonomic designation lll. Receipt and acceptance This International Depositary Authority accepts the microorganism identified under I above, which was received by it on (date of original deposit) 23 november 1993 IV. International Depositary Authority Belgian Coordinated Collections of Microorganisms (BCCM) Mycothèque de l'Université Catholique de Louvain (MUCL) Place Croix du Sud 3 B-1 348 Louvain-la-Neuve Belgium Signature(s) of person(s) having the power to represent the International Depositary Authority or of authorized official(s):
Plufessor G.L. Hennebert r '-2,4- ~
Date : 8 december 1993 .,
Claims (10)
1 - Lipolytic enzymes naturally produced by a fungus of the species Fusarium solanii.
2 - The lipolytic enzymes according to claim 1, characterized in that they are naturally produced by a fungus of the species Fusarium solanii var.
minus.
minus.
3 - The lipolytic enzymes according to claim 2, characterized in that they are naturally produced by Fusarium solanii var. minus T.92.637/1.
4 - The lipolytic enzymes according to claim 1, 2 or 3, characterized in that the lipolytic enzymes include a lipase.
5 - The lipolytic enzymes according io claim 4, characterized in that the lipase has an isoelectric point of about 5.2 and an apparent molecular weight of about 60 kDa.
6 - The lipolytic enzymes according to claim 4, characterized in that the lipase has an isoelectric point of about 6.9 and an apparent molecular weight of about 30 kDa.
7 - The lipolytic enzymes according to claim 2 or 3, characterized in that the lipolytic enzymes include a cutinase.
8 - The lipolytic enzymes according to claim 7, characterized in that the cutinase has an isoelectric point of about 7.2 and an apparent molecular weight of about 22 kDa.
9 - A biologically pure culture of Fusarium solanii var. minus T.92.637/1 and mutants thereof.
10 - A detergent composition including at least one lipolytic enzyme according to claim 1.
11 - A method for removing fat stains from fabrics, said method comprising washing said fabrics with a detergent composition according to
10 - A detergent composition including at least one lipolytic enzyme according to claim 1.
11 - A method for removing fat stains from fabrics, said method comprising washing said fabrics with a detergent composition according to
claim 10.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9425109.7 | 1994-12-13 | ||
| GB9425109A GB2296011B (en) | 1994-12-13 | 1994-12-13 | Novel fusarium isolate and lipases, cutinases and enzyme compositions derived therefrom |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2208223A1 true CA2208223A1 (en) | 1996-06-20 |
Family
ID=10765854
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002208223A Abandoned CA2208223A1 (en) | 1994-12-13 | 1995-12-02 | Fusarium isolate and lipases, cutinases and enzyme compositions derived therefrom |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US5990069A (en) |
| EP (1) | EP0799306A1 (en) |
| JP (1) | JPH11502401A (en) |
| AU (1) | AU706474B2 (en) |
| BR (1) | BR9510009A (en) |
| CA (1) | CA2208223A1 (en) |
| FI (1) | FI972496A (en) |
| GB (1) | GB2296011B (en) |
| NZ (1) | NZ298017A (en) |
| WO (1) | WO1996018729A1 (en) |
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| US6936289B2 (en) | 1995-06-07 | 2005-08-30 | Danisco A/S | Method of improving the properties of a flour dough, a flour dough improving composition and improved food products |
| US6103505A (en) * | 1996-12-09 | 2000-08-15 | Novo Nordisk A/S | Method for reducing phosphorus content of edible oils |
| DE69806586T2 (en) | 1997-04-09 | 2003-03-27 | Danisco As Kopenhagen Koebenha | IMPROVED METHOD FOR THE PRODUCTION OF FLOOD PASTA AND PRODUCTS MADE FROM SUCH PASTA USING GLYZERINE OXIDASE |
| EP1066374B1 (en) * | 1998-02-27 | 2006-05-31 | Novozymes A/S | Amylolytic enzyme variants |
| ES2188190T5 (en) | 1998-07-21 | 2007-11-16 | Danisco A/S | FOOD PRODUCT. |
| US7312062B2 (en) | 1998-11-27 | 2007-12-25 | Novozymes A/S | Lipolytic enzyme variants |
| DK2290059T3 (en) | 1998-11-27 | 2016-02-22 | Novozymes As | Lipolytic enzyme VERSIONS |
| US6933140B1 (en) | 1999-11-05 | 2005-08-23 | Genencor International, Inc. | Enzymes useful for changing the properties of polyester |
| US7226770B2 (en) | 2000-04-28 | 2007-06-05 | Novozymes A/S | Lipolytic enzyme variant |
| EP2119773A1 (en) | 2000-06-26 | 2009-11-18 | Novozymes A/S | Lipolytic enzymes from strains of fusarium and acremonium |
| US6506588B2 (en) | 2000-06-26 | 2003-01-14 | Novozymes, A/S | Lipolytic enzymes |
| EP1301080B1 (en) | 2000-07-06 | 2011-09-14 | Novozymes A/S | Method of preparing a dough, or a baked product made from a dough, with addition of lipolytic enzymes |
| AU2002219020B2 (en) | 2001-01-10 | 2007-05-24 | Novozymes A/S | Thermostable lipolytic enzyme variant |
| ES2284897T3 (en) | 2001-05-18 | 2007-11-16 | Danisco A/S | PROCEDURE FOR THE PREPARATION OF A MASS WITH AN ENZYME. |
| US6645749B2 (en) | 2001-05-25 | 2003-11-11 | Novozymes A/S | Lipolytic enzyme |
| US7050821B2 (en) * | 2002-10-29 | 2006-05-23 | Matsushita Electric Industrial Co., Ltd. | Digital radio communications apparatus |
| DE602004030000D1 (en) | 2003-01-17 | 2010-12-23 | Danisco | PROCESS FOR IN-SITU-PRODUCTION OF AN EMULSIFIER IN A FOODSTUFF |
| US20050196766A1 (en) | 2003-12-24 | 2005-09-08 | Soe Jorn B. | Proteins |
| US7955814B2 (en) | 2003-01-17 | 2011-06-07 | Danisco A/S | Method |
| EP1625202A4 (en) * | 2003-05-12 | 2010-10-20 | Genencor Int | Novel lipolytic enzyme lip1 |
| US20070213518A1 (en) * | 2003-05-12 | 2007-09-13 | Jones Brian E | Novel Lipolytic Enzyme Lip2 |
| WO2004101760A2 (en) | 2003-05-12 | 2004-11-25 | Genencor International, Inc. | Novel lipolytic enzyme elip |
| GB0716126D0 (en) | 2007-08-17 | 2007-09-26 | Danisco | Process |
| US7718408B2 (en) | 2003-12-24 | 2010-05-18 | Danisco A/S | Method |
| US7906307B2 (en) | 2003-12-24 | 2011-03-15 | Danisco A/S | Variant lipid acyltransferases and methods of making |
| GB0405637D0 (en) | 2004-03-12 | 2004-04-21 | Danisco | Protein |
| DK1791933T3 (en) | 2004-07-16 | 2011-09-05 | Danisco | Process for enzymatic boiling of oil |
| EP1833918B1 (en) | 2004-12-23 | 2011-09-28 | Dow Corning Corporation | Crosslinkable saccharide-siloxane compositions, and networks, coatings and articles formed therefrom |
| CN101217934B (en) | 2005-05-23 | 2013-01-02 | 陶氏康宁公司 | Personal care compositions comprising saccharide-siloxane copolymers |
| JP5185259B2 (en) | 2006-05-23 | 2013-04-17 | ダウ・コーニング・コーポレイション | Novel silicone film forming agent for active ingredient delivery |
| CA2673954C (en) | 2007-01-25 | 2015-09-15 | Danisco A/S | Production of a lipid acyltransferase from transformed bacillus licheniformis cells |
| FI120835B (en) * | 2007-07-10 | 2010-03-31 | Valtion Teknillinen | New esters and their use |
| EP2197887A1 (en) * | 2007-09-28 | 2010-06-23 | Dow Corning Corporation | Organosiloxanes containing ester derivatives of ascorbic acid |
| BR112012018822A2 (en) | 2009-12-21 | 2019-09-24 | Danisco Us Inc | surfactants that optimize the cleaning of lipase-treated lipase-treated stains |
| BR112012017060A2 (en) | 2009-12-21 | 2016-11-29 | Danisco Us Inc | "Thermobifide lipase-containing detergent compositions, method for hydrolyzing a lipid present in a dirt or stain on a surface and method for performing a transesterification reaction |
| CN102712878A (en) | 2009-12-21 | 2012-10-03 | 丹尼斯科美国公司 | Detergent compositions containing bacillus subtilis lipase and methods of use thereof |
| CN102712880A (en) | 2009-12-21 | 2012-10-03 | 丹尼斯科美国公司 | Detergent compositions containing geobacillus stearothermophilus lipase and methods of use thereof |
| WO2011150157A2 (en) | 2010-05-28 | 2011-12-01 | Danisco Us Inc. | Detergent compositions containing streptomyces griseus lipase and methods of use thereof |
| JP5997155B2 (en) | 2010-08-23 | 2016-09-28 | ダウ コーニング コーポレーションＤｏｗ Ｃｏｒｎｉｎｇ Ｃｏｒｐｏｒａｔｉｏｎ | Sugar siloxanes that are stable in an aqueous environment and methods of preparing and using such sugar siloxanes |
| EP2627777A1 (en) | 2010-10-13 | 2013-08-21 | Novozymes A/S | Processing of oils and fats |
| US9175256B2 (en) | 2010-12-23 | 2015-11-03 | Exxonmobil Research And Engineering Company | Production of fatty acids and fatty acid derivatives by recombinant microorganisms expressing polypeptides having lipolytic activity |
| JP6027092B2 (en) | 2011-04-08 | 2016-11-16 | ダニスコ・ユーエス・インク | Composition |
| BR112014004444A2 (en) | 2011-08-31 | 2017-03-21 | Danisco Us Inc | compositions and methods comprising a lipolytic enzyme variant |
| EP2794866A1 (en) | 2011-12-22 | 2014-10-29 | Danisco US Inc. | Compositions and methods comprising a lipolytic enzyme variant |
| AU2013328953A1 (en) | 2012-10-12 | 2015-03-26 | Danisco Us Inc. | Compositions and methods comprising a lipolytic enzyme variant |
| MX371497B (en) | 2013-07-19 | 2020-01-31 | Danisco Us Inc | Compositions and methods comprising a lipolytic enzyme variant. |
| CN108431217B (en) | 2015-12-01 | 2022-06-21 | 诺维信公司 | Method for producing lipase |
| WO2020046613A1 (en) | 2018-08-30 | 2020-03-05 | Danisco Us Inc | Compositions comprising a lipolytic enzyme variant and methods of use thereof |
| WO2021146255A1 (en) | 2020-01-13 | 2021-07-22 | Danisco Us Inc | Compositions comprising a lipolytic enzyme variant and methods of use thereof |
| KR20240027619A (en) | 2021-06-30 | 2024-03-04 | 헨켈 아게 운트 코. 카게아아 | Compositions with improved moisture management performance |
| WO2023278297A1 (en) | 2021-06-30 | 2023-01-05 | Danisco Us Inc | Variant lipases and uses thereof |
| CN117580940A (en) | 2021-06-30 | 2024-02-20 | 汉高股份有限及两合公司 | Cleaning compositions comprising lipolytic enzyme having polyesterase activity |
| CN117580939A (en) | 2021-06-30 | 2024-02-20 | 汉高股份有限及两合公司 | Cleaning compositions having improved anti-dusting and/or anti-pilling properties |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK289083A (en) * | 1983-06-23 | 1984-12-24 | Novo Industri As | LIPASE, PROCEDURE FOR PREPARING THEREOF AND ITS APPLICATION |
| WO1990009446A1 (en) * | 1989-02-17 | 1990-08-23 | Plant Genetic Systems N.V. | Cutinase |
| GB9216387D0 (en) * | 1992-07-31 | 1992-09-16 | Unilever Plc | Enzymatic detergent compositions |
| AU5699994A (en) * | 1992-12-18 | 1994-07-19 | Unilever Plc | Modified cutinases, dna, vector and host |
| DK154292D0 (en) * | 1992-12-23 | 1992-12-23 | Novo Nordisk As | NEW ENZYM |
| EP0802981A1 (en) * | 1992-12-23 | 1997-10-29 | Unilever N.V. | Modified cutinases, dna, vector and host |
-
1994
- 1994-12-13 GB GB9425109A patent/GB2296011B/en not_active Expired - Fee Related
-
1995
- 1995-12-02 AU AU43405/96A patent/AU706474B2/en not_active Ceased
- 1995-12-02 CA CA002208223A patent/CA2208223A1/en not_active Abandoned
- 1995-12-02 EP EP95942072A patent/EP0799306A1/en not_active Withdrawn
- 1995-12-02 WO PCT/EP1995/004799 patent/WO1996018729A1/en not_active Application Discontinuation
- 1995-12-02 BR BR9510009A patent/BR9510009A/en not_active Application Discontinuation
- 1995-12-02 US US08/849,824 patent/US5990069A/en not_active Expired - Lifetime
- 1995-12-02 JP JP8518229A patent/JPH11502401A/en active Pending
- 1995-12-02 NZ NZ298017A patent/NZ298017A/en unknown
-
1997
- 1997-06-12 FI FI972496A patent/FI972496A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0799306A1 (en) | 1997-10-08 |
| WO1996018729A1 (en) | 1996-06-20 |
| AU4340596A (en) | 1996-07-03 |
| GB2296011B (en) | 1999-06-16 |
| GB2296011A (en) | 1996-06-19 |
| GB9425109D0 (en) | 1995-02-08 |
| FI972496A0 (en) | 1997-06-12 |
| NZ298017A (en) | 1999-03-29 |
| JPH11502401A (en) | 1999-03-02 |
| BR9510009A (en) | 1997-10-21 |
| AU706474B2 (en) | 1999-06-17 |
| US5990069A (en) | 1999-11-23 |
| FI972496A (en) | 1997-08-12 |
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