WO1996036700A1

WO1996036700A1 - An enzyme with exochitinase activity

Info

Publication number: WO1996036700A1
Application number: PCT/DK1996/000216
Authority: WO
Inventors: Henriette Draborg; Stephan Christgau
Original assignee: Novo Nordisk A/S
Priority date: 1995-05-16
Filing date: 1996-05-15
Publication date: 1996-11-21
Also published as: AU5685396A

Abstract

The invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting exochitinase activity, which DNA sequence comprises the DNA sequence shown in SEQ ID No. 1 or SEQ ID No. 3, or analogues of the DNA sequences encoding polypeptides which are derived from Saccharomyces cerevisiae DSM No. 9944 or Saccharomyces cerevisiae DSM No. 9945. Further, the invention relates to a method of producing said enzyme, an enzyme preparation containing said enzyme, and the use of said exochitinase or said enzyme preparation for a number of purposes. Also claimed is the use of the DNA construct of the invention for producing transgenic plants. Finally, an isolated substantially pure culture of the deposited microorganisms is claimed.

Description

AN ENZYME WITH EXOCHITINASE ACTIVITY

FIELD OF INVENTION

The present invention relates to an enzyme with exochitinase activity, a DNA construct encoding said enzyme, a method of producing said enzyme, and an enzyme preparation containing said enzyme.

Further the invention relates to the use of said exochitinase or said enzyme preparation for a number of purposes.

BACKGROUND OF THE INVENTION

Considerable amount of chitin is found in the cells wall of most true fungal cells, in the exoskeleton of insects and crustaceans, and in nematodes (Cabib E., (1987) Adv. Enzymol., 59, 59-101; Gooday G., (1990), Microbial Ecol., 10, 397-431).

Native occurring chitin consist of a chemically stable insoluble polymer substance composed of β-l,4-N-acetyl glucosamine molecules of varying length, stabilized by hydrogen bonds to a higher ordered crystalline structure (Cabib E., (1987), Adv. Enzymol., 59, 59-101).

The polymer molecules of chitin are bound by β-1,4 linkages, and are in fungal cell walls often associated with β-1,3 glucan or β-1,6 glucan, polymers of glucose with β-1,3 and β-1,6 link- ages, as well as various intrinsic call wall components

Chitin is known to be hydrolysed by chitinases, including exo- and endochitinases, which is present in most fungi, yeasts, plants and certain procaryotes (Cabib E. , (1987), Adv. Enzymol., 59, 59-101; Gooday G. , (1990), Microbial Ecol., 10, 397-431) . Exochitinases are enzymes that exolytically hydrolyse the β- 1,4-linkage between two consecutive N-acetylglucosamines from the non-reducing end of chitin.

Exochitinases are also referred to as chitobiosidases or β-N- acetylhexosaminidases (E.C. 3.2.1.52, Enzyme Nomenclature, Academic Press, Inc., 1992).

Endochitinases (E.C. 3.4.1.14) are enzymes which randomly hydrolyse N-acetyl-β-D-glucosaminide 1,4-β-linkages of chitin and chitodextrins.

Chitinases, are thought to play an important role in the cell division and differentiation of fungi, and in the mycoparasitic activity displayed by several fungi, such as Gliocladium virens, Aphanocladium album and Trichoderma harzianum (De La

Cruz et al. (1992) Eur. J. Biochem. , 206, 859-867; Blaiseau and

Lafay, (1992), Elsevier science publisher B.V., 243-248; Di Pietro et al., (1993), Mol. Plant Pathology 83, 308-313).

Although many fungal chitinases have been reported, only few of them have been cloned and characterized at molecular level

(Harman et al., (1993), Mol. Plant Pathology 83, 313-318;

Blaiseau and Lafay, (1992), supra; Gracia, (1994), Current Genetics 27, 83-89.

A number of readily available commercial enzyme products useful in the enzymatic lysis of fungal cells comprise chitinase. Beside chitinase(s) such products normally also comprise multiple enzymatic activities, e .g. including β-1,3- and β-1,6- glucanase, cellulase, protease, mannase and other enzymes capable of degrading cell wall components.

WO 92/22314 (Cornell Research Foundation, INC) describes two chitinases from Trichoderma harzianum PI (ATCC 74058) which inhibit chitin containing fungus and insects. The first is an endochitinase having a molecular weight of 36 kDa and an isoelectric point of about 5.3. The second is an exochitinase having a molecular weight of 36 kDa and an isoelectric point of about 4.4.

WO 94/24288 and WO 94/02598 (Cornell Research Foundation, INC) discloses two chitinases from Trichoderma harzianum PI (ATCC 74058) which inhibit chitin containing fungus and insects. The first is an endochitinase and the other is an chitobiosidase. Both have molecular weights of 40 kDa and isoelectric points of about 3.9.

EP 440.304 concerns plants exhibiting a relative overexpression of at least one gene encoding intracellular chitinase and intra- or extracellular β-1,3 glucanase. Further, the recombinant polynucleotides are disclosed.

Comments to prior art

In general enzyme preparations containing chitinases also contain a number of other enzyme activities. This may in certain cases be a drawback.

Said drawback may be remedied by using single-component enzymes (i.e. substantially without any side activity) exhibiting chitinase activity.

Consequently there exists a need for providing novel chitinases, preferably in single-component form, which may be used for applications where a single or dominating chitinolytic activity is desirable. SUMMARY OF THE INVENTION

The present inventors have now surprisingly succeeded in iso¬ lating and characterizing two DNA sequences which encode enzymes exhibiting exochitinase activity, thereby making it possible to prepare single-component exochitinases.

Accordingly, in a first aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhi¬ biting exochitinase activity, which DNA sequence a) comprises the DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3, or b) comprises an analogue of the DNA sequence shown in SEQ ID no. 1 or SEQ ID no 3, which i) is homologous with the DNA sequences shown in SEQ ID no. 1 or SEQ ID no. 3, and/or ii) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID no. 1 or SEQ ID no. 3, and/or iii) encodes a polypeptide which is homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3, and/or iv) encodes a polypeptide which is immunologically reactive with an antibody raised against a purified exochitinase shown in SEQ ID no. 2 derived from Saccharomyces cerevisiae DSM no. 9944 or against a purified Exochitinase shown in SEQ ID no. 4 derived from Saccharomyces cerevisiae DSM no. 9945.

In a second aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting exochitinase activity, which DNA sequence comprises at least a partial sequence of the DNA sequence shown in SEQ ID no. 1 or SEQ ID no. 3. In the present context, the "analogue" of the DNA sequence shown in SEQ ID no. 1 or SEQ ID no. 3 is intended to indicate any DNA sequence encoding an enzyme exhibiting exochitinase activity, which has the properties i)-vi). The analogous DNA sequence

- may be isolated from any organism producing the enzyme with exochitinase activity on the basis of a partial DNA sequence comprised in the DNA sequence shown in SEQ ID no. 1 or SEQ ID no. 3, e .g. using the procedures described herein, and thus, e .g. be an allelic or species variant of the DNA sequence shown herein,

- may be constructed on the basis of a partial DNA sequence comprised in the DNA sequences shown in SEQ ID no. 1 or SEQ ID no. 3, e .g. by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the exochitinase encoded by the DNA sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. However, in the latter case amino acid changes are preferably of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding or activity of the protein, small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal ex¬ tensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small exten- sion that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain. See in general Ford et al., (1991), Protein Expression and Purification, 2, 95-107. Examples of conservative substitutions are within the group of basic amino acids (such as arginine, lysine, histi- dine) , acidic amino acids (such as glutamic acid and aspartic acid) , polar amino acids (such as glutamine and asparagine) , hydrophobic amino acids (such as leucine, isoleucine, valine) , aromatic amino acids (such as phenylalanine, tryptophan, ty¬ rosine) and small amino acids (such as glycine, alanine, se- rine, threonine, methionine) .

It will be apparent to persons skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active poly¬ peptide. Amino acids essential to the activity of the poly¬ peptide encoded by the DNA construct of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis

(Cunningham and Wells, (1989), Science, 244, 1081-1085). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological (i.e. exochitinolytic) activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labelling. See, for example, de Vos et al., (1992), Science, 255, 306-312; Smith et al., (1992), J. Mol. Biol., 224, 899-904; Wlodaver et al., (1992) FEBS Lett., 309, 59-64.

It will be understood that any partial sequences comprised in the DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3 may be used for isolating the entire DNA sequence encoding the enzyme with exochitinase activity, e .g. the DNA sequence shown in SEQ ID No. 1. DNA sequences encoding at least about 6 amino acids of the sequence shown in SEQ ID No. 2 or SEQ ID no. 4 are comtemplated as partial sequences according to the invention. The term "analogue" is intended to include said entire DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3, or parts thereof. The amino acid sequences (as deduced from the DNA sequence shown in SEQ ID No. 1 and SEQ ID no. 3) are shown in SEQ ID No. 2 and SEQ ID no. 4. The homology referred to in i) above is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, p. 443-453) . Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the DNA sequence exhibits a degree of identity of at least 70%, preferably at least 80%, especially at least 90%, with the coding region of the DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3.

The hybridization referred to in ii) above is intended to indicate that the analogous DNA sequence hybridizes to the same probe as the DNA sequence encoding the exochitinase under certain specified conditions which are described in detail in the Materials and Methods section hereinafter.

Normally, the analogous DNA sequence is highly homologous to the DNA sequence such as at least 70% homologous to the DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3 encoding exochitinases of the invention, such as at least 75%, at least 80%, at least 85%, at least 90% or even at least 95% homologous to said DNA sequence

The homology referred to in iii) above is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, p. 443-453). Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the DNA sequence exhibits a degree of identity of at least 70%, preferably at least 80%, especially at least 90%, with the coding region of the DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3.

The term "derived from" in connection with property vi) above is intended not only to indicate an exochitinase produced by the above mentioned deposited strain Saccharomyces cerevisiae DSM no. 9944 or DSM no. 9945, but also an exochitinase encoded by a DNA sequence isolated from the deposited strain DSM no. 9944 or DSM no. 9945 and produced in a host organism transformed with said DNA sequence. The immunological reacti¬ vity may be determined by the method described in the Materials and Methods section below.

In further aspects the invention relates to an expression vec¬ tor harbouring a DNA construct of the invention, a cell compri¬ sing the DNA construct or expression vector and a method of producing an enzyme exhibiting exochitinase activity which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.

In a still further aspect the invention relates to an enzyme exhibiting exochitinase activity, which enzyme a) is encoded by a DNA construct of the invention b) produced by the method of the invention, and/or c) is immunologically reactive with an antibody raised against a purified exochitinase shown in SEQ ID No. 2 derived from Saccharomyces cerevisiae DSM no. 9944 or against a purified exochitinase shown in SEQ ID no. 4 derived from Saccharomyces cerevisiae DSM 9945.

In a still further aspect, the present invention relates to an enzyme preparation useful for the degradation or modification of fungal, invertebrate, or nematode cell wall components, said preparation being enriched with an enzyme exhibiting exochitinase activity as described above. The invention also relates to the use of an exochitinase of the invention or an enzyme preparation of the invention comprising an exochitinase of the invention for plant protection and pharmaceutical purposes.

Finally the invention relates to an isolated substantially pure culture of the deposited strain of Saccharomyces cerevisiae DSM no. 9944 or DSM no. 9945, transformed with a plasmid-DNA comprising the DNA sequences shown in SEQ ID No. 1 and SEQ ID no. 3, respectively or a partial DNA sequence of these two sequences.

DETAILED DESCRIPTION OF THE INVENTION

The DNA sequence of the invention encoding an enzyme exhibiting exochitinase activity may be isolated by a general method involving - cloning, in suitable vectors, a cDNA library from e .g. a strain of Saccharomyces sp. , Aspergillus sp. , Trichoderma sp., Penicillium sp., Fusarium sp., Gliocladium sp.,

Aphanocladium sp. , or Humicola sp.,

- transforming suitable yeast host cells with said vectors, - culturing the host cells under suitable conditions to ex¬ press any enzyme of interest encoded by a clone in the DNA library, screening for positive clones by determining any exochitinase activity of the enzyme produced by such clones, and

- isolating the enzyme encoding DNA from such clones.

The general method is further disclosed in WO 93/11249 the con¬ tents of which are hereby incorporated by reference. A more de- tailed description of the screening method is given in Example 2 below. Two isolates of Saccharomyces cerevisiae transformed with the expression plasmid pYES 2.0 (Invitrogen) , comprising the cDNA sequences, shown in SEQ ID No. 1 and SEQ ID No 3, respectively, encoding exochitinases of the invention, have been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-3300 Braunschweig, Federal Republic of Germany, (DSM) , for the purposes of patent procedure on the date indicated below. DSM being an international depository under the Budapest Treaty affords permanence of the deposit in accordance with rule 9 of said treaty.

Deposit date : 27.04.95 Depositor's ref.: NN14684 CBS designation : DSM No. 9944

Deposit date : 27.04.95

Depositor's ref.: NN14685

CBS designation : DSM No. 9945

The two plasmid-DNA isolates of the fungus Saccharomyces cerevisiae DSM no. 9944 (NN14684) and DSM no. 9945 (NN14685) , respectively have been deposited under conditions that assure that access to the isolated yeast will be available during the pendency of this patent application to one determined by the commissioner of Patents and Trademarks to be entitled thereto under 37 CF.R. § 1.14 and 35 U.S.C § 122.

Further the above mentioned depositions of the yeast Saccharomyces cerevisiae DSM no. 9944 and DSM no. 9945 have been done to fulfil the requirements of a European patent applications under Rule 28 of the European Patent Convention.

The deposits represent substantially pure cultures of the two isolated yeast. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of the deposits does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

A DNA sequence coding for the enzyme exhibiting exochitinase activity can for instance be isolated from the above mentioned deposited strains as described in Example 1.

Further said DNA sequence may be isolated by screening a cDNA library of e .g. the above mentioned group of fungi, and selecting for clones expressing the appropriate enzyme activity

(i.e. exochitinase activity as defined by the ability of the enzyme to hydrolyse the β-l,4-linkage of a suitable substrate, such as the synthetic chitin substrate 4-methylumbelliferyl N- acetylglucosaminide (MUF-GlcNAc) , cf . the Materials and Methods section herein after) . The appropriate DNA sequence may then be isolated from the clone by standard procedures.

It is expected that a DNA sequence coding for a homologous en¬ zyme, i.e. an analogous DNA sequence, is obtainable from other microorganisms. For instance, the DNA sequence may be derived by screening a cDNA library of another microorganism, such as in particular a fungus, such as a strain of an Aspergillus sp., in particular a strain of A. aculeatus or A. niger, a strain of another Trichoderma sp. , in particular a strain of T. reesie, T. viride , T. longibrachiatum , T. harzianum or T. koningii or a strain of a Fusarium sp., in particular a strain of F. oxysporum, or a strain of Gliocladium sp. , in particular Gliocladium virens , or a strain of Aphanocladium, in particular Aphanocladium album, or a strain of a Humicola sp., or a strain of Beauveria sp., in particular Beauveria bassiana, or a strain of Metarhizium sp. , in particular Metarhizium anisopliae, or a strain of Mucor sp. , in particular Mucor rouxii , or a mutant thereof capable of producing a compound of the invention.

Alternatively, the DNA coding for an exochitinase of the invention may, in accordance with well-known procedures, conveniently be isolated from DNA from a suitable source, such as any of the above mentioned organisms, by use of synthetic oligonucleotide probes prepared on the basis of a DNA sequence disclosed herein. For instance, a suitable oligonucleotide probe may be prepared on the basis of the nucleotide sequences shown in SEQ ID No. 1 or SEQ ID no. 3, or a partial sequence thereof, or the amino acid sequences shown in SEQ ID No. 2 or SEQ ID no. 4, or any suitable subsequence thereof.

The DNA sequence may subsequently be inserted into a recom- binant expression vector. This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an auto¬ nomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is indepen¬ dent of chromosomal replication, e .g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding the exochitinase should be operably connected to a suitable promoter and terminator sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. The procedures used to ligate the DNA sequences coding for the exochitinase, the promoter and the terminator, respectively, and to insert them into suitable vectors are well known to persons skilled in the art (cf., for instance, Sambrook et al., (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY) .

The host cell which is transformed with the DNA sequence encoding the enzyme of the invention is preferably an eukaryotic cell, in particular a fungal cell such as a yeast or filamentous fungal cell. In particular, the cell may belong to a species of Trichoderma , preferably Trichoderma harzianum or Trichoderma reesie, or a species of Aspergillus, preferably Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se . The use of Aspergillus as a host microorganism is described in EP 238 023 (of Novo Nordisk A/S) , the contents of which are hereby incorporated by reference. The host cell may also be a yeast cell, e.g. a strain of Saccharomyces, in particular Saccharomyces cerevisiae , Saccharomyces kluyveri or Saccharomyces uvarum, a strain of Schizosaccharomyces sp. , such as Schizosaccharomyces pombe, a strain of Hansenula sp. Pichia sp., Yarrowia sp. such as Yarrowia lipolytica , or Kluyveromyces sp. such as Kluyveromyces lactis .

In a still further aspect, the present invention relates to a method of producing an enzyme according to the invention, wherein a suitable host cell transformed with a DNA sequence encoding the enzyme is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.

The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed exochitinase may conveniently be secreted into the culture medium and may be recovered there from by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

In a still further aspect, the present invention relates to an enzyme preparation useful for the degradation or modification of fungal cell wall components, said preparation being enriched in an enzyme exhibiting exochitinase activity as described above .

In the present context, the term "enriched" is intended to indicate that the exochitinase activity of the enzyme preparation has been increased, e .g. with an enrichment factor of at least 1.1, conveniently due to addition of an enzyme of the invention prepared by the method described above.

Alternatively, the enzyme preparation enriched in an enzyme exhibiting exochitinase activity may be one which comprises an enzyme of the invention as the major enzymatic component, e .g. a single-component enzyme preparation.

The enzyme preparation may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry preparation. For instance, the enzyme preparation may be in the form of a granulate or a microgranulate. The enzyme to be included in the preparation may be stabilized in accordance with methods known in the art.

The enzyme preparation of the invention may, in addition to an exochitinase of the invention, contain one or more other fungal, invertebrate, such as crustacean, and/or nematode cell wall degrading enzymes, for instance those with proteolytic, β- glucanolytic, mannanolytic, chitinolytic activities, such as protease, β-glucanase, β-mannosidase, mannanase, β-glucosidase, endochitinase, and mannan acetyl esterase. The additional enzyme(s) may be producible by means of a microorganism belonging to the genus Aspergillus, preferably Aspergillus niger, Aspergillus aculeatus, Aspergillus awamori or Aspergil¬ lus oryzae, or Trichoderma sp..

Examples are given below of preferred uses of the enzyme prepa¬ ration of the invention. The dosage of the enzyme preparation of the invention and other conditions under which the prepara¬ tion is used may be determined on the basis of methods known in the art. The enzyme preparation according to the invention may be used as an agent for degradation or modification of fungal, invertebrate and/or nematode cell walls, or for making protoplasts from fungi.

Further exochitinases of the invention may advantageously be used for protecting plants against nematode eggs and generally against fungal infection, e .g. by coating the seeds, or by spraying the plants.

In connection with combating fungal microorganisms on plants a DNA sequence encoding the exochitinase of the invention may be used for producing transgenic plants, by introducing said DNA sequence into a plant. This may increase the plants resistance against microorganisms of fungal origin.

Another advantageous use of exochitinases of the invention include modification of chitin for use for pharmaceutical purposes, e .g. for wound dressing.

Finally the invention relates to substantially pure cultures of the deposited microorganisms Saccharomyces cerevisiae DSM no. 9944 and DSM no. 9945, which comprises the above described DNA construct of the invention, containing a DNA sequence encoding an exochitinase of the invention.

The invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention as claimed.

MATERIALS AND METHODS

Deposited organisms:

Saccharomyces cerevisiae DSM no. 9944 transformed with the DNA sequence shown in SEQ ID No. 1, encoding the exochitinase of the invention, comprised in the expression plasmid pYES 2.0.

Saccharomyces cerevisiae DSM no. 9945 transformed with the DNA sequence shown in SEQ ID No. 3, encoding the exochitinase of the invention, comprised in the expression plasmid pYES 2.0.

Plasmids: pYES 2.0 (Invitrogen) pClEXCl: SEQ ID. no. 1 sequence in pYES 2.0 pClEXC2: SEQ ID. no. 3 sequence in pYES 2.0

Isolation of the the DNA sequence shown in SEQ ID no. l and SEQ ID no. 3

The yeast expression vectors of the type pYES 2.0 containing the exochitinase cDNA sequences shown in SEQ ID no. 1 and SEQ ID no. 3, respectively, can be isolated from the deposited organism Saccharomyces cerevisiae DSM no. 9944 and DSM 9945, restectively by extraction of plasmid cDNA by methods known in the art.

The deposited organisms may be cultured on agar plates containing SC + 2% galactose and incubated at 30°C for 3-5 days as described below.

Hybridization conditions (to be used in evaluating property i) of the DNA construct of the invention) :

Suitable conditions for determining hybridization between an oligonucleotide probe and an "analogous" DNA sequence involves presoaking of the filter containing the DNA sequences to hybri¬ dize in 5xSSC and prehybridizing the sequences for 1 h at ~55°C in a solution of 2xSSC, SxDenhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 μg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplement- ed with 50 μCi 32-P-dCTP labelled probe for 18 h at "55°C fol¬ lowed by washing in 2xSSC (2x15 minutes), 2xSSC, 0.2% SDS (1x30 minutes), 0.2xSSC, 0.5% SDS (1x30 minutes), 2xSSC (2x15 minutes) at 55°C

A suitable oligonucleotide probe to be used in the hybridiza- tion may be prepared on the basis of the DNA sequences shown in

SEQ ID No. 1 and SEQ ID no. 3, respectively, or partial sequences thereof.

Immunological cross-reactivity: Antibodies to be used in deter- mining immunological cross-reactivity may be prepared by use of a purified exochitinase. More specifically, antiserum against the exochitinase of the invention may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al. in: A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 23, or A. Johnstone and R. Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, (1982) (more specifically p. 27-31) . Purified immunoglobulins may be obtained from the antisera, for example by salt precipitation ((NH_ι)₂ SO₄) , followed by dialysis and ion exchange chromatography, e .g. on DEAE-Sephadex. Immunochemical characterization of proteins may be done either by Outcherlony double-diffusion analysis (O. Ouchterlony in: Handbook of Experimental Immunology (D.M. Weir, Ed.), Blackwell Scientific Publications, (1967) , p. 655-706) , by crossed immunoelectrophoresis (N. Axelsen et al., supra. Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsen et al.. Chapter 2) .

Media

SC-URA: 90 ml 10 x Basal salt, 22.5 ml 20% casamino acids, 9 ml 1% tryptophan, H0 ad 806 ml, autoclaved, 3.6 ml 5% threonine and 90 ml 20% glucose or 20% galactose added.

SC-H broth: 7.5 g/1 yeast nitrogen base without amino acids, 11.3 g/1 succinic acid, 6.8 g/1 NaOH, 5.6 g/1 casamino acids without vitamins, 0.1 g/1 tryptophan. Autoclaved for 20 min. at 121°C After autoclaving, 10 ml of a 30% galactose solution, 5 ml of a 30% glucose solution and 0.4 ml of a 5% threonine solution were added per 100 ml medium.

SC-H agar: 7.5 g/1 yeast nitrogen base without amino acids, 11.3 g/1 succinic acid, 6.8 g/1 NaOH, 5.6 g/1 casamino acids without vitamins, 0.1 g/1 tryptophan, and 20 g/1 agar (Bacto) . Autoclaved for 20 min. at 121°C After autoclaving, 55 ml of a 22% galactose solution and 1.8 ml of a 5% threonine solution were added per 450 ml agar.

4-methylumbelliferyl N-acetylglucosaminide (MUF-GlcNAc) (Sigma, USA)

MUF-N,N'-diacetylchitobioside (MUF-(GlcNAc)₂) (Sigma, USA)

MUF-N,N' ,N' '-tridiacetylchitotrioside (MUF-(GlcNAc)₃) (Sigma, USA)

Qiagen purified plasmid DNA (Qiagen, USA) ,

Sequenase®kit (U.S.Biochemical corp., USA)

Remazol Brilliant Violet colloidal chitin (Sigma, USA)

Random hexanucleotide primers (Gibco BRL, USA)

EXAMPLE 1

Characterization of enzyme from yeast

Saccharomyces cerevisiae DSM No. 9944 and DSM no. 9945 were cultured separetly on SC-glucose plates for 3-4 days at 40°C The plates were replicated onto a set of four selective agar plates containing 2% galactose. One of the plates was also supplemented with 0.2% Remazol Brilliant Violet colloidal chitin.

After incubation at 30°C for three days SC-agar plates with 2% galactose were overlayerd with 10 ml 1% agarose, 0.1 M citric acid/sodium citrate buffer, pH 5.0, containing 1 mg of MUF- (GlcNAc) , MUF-(GlcNAc)₂ or MUF-(GlcNAc)₃ each.

The release of 4-methylumbelliferyl by chitinases was visualized under UV-light.

The recombinant yeast harbouring the plasmids pClEXCl showed activity on MUF-GlcNAc immediately after the agarose overlayer was spread over the yeast colonies, whereas activities on MUF- (GlcNAc)₂ and MUF-(GlcNAc)₃ were visualized only after 5 and 10 minutes of incubation, respectively. By comparison, .the recombinant yeast strain showed no activity on the hydrated colloidal chitin.

The recombinant yeast harbouring the plasmids pClEXC2 showed activity on MUF-GlcNAc immediately after the agarose overlayer was spread over the yeast colonies, whereas activities on MUF- (GlcNAc)₂ and MUF-(GlcNAc)₃ were visualized only after 5 and 10 minutes of incubation, respectively. By comparison, the recombinant yeast strain showed no activity on the hydrated colloidal chitin.

EXAMPLE 2

Construction and screening of the cDNA library

Total RNA was prepared from frozen, powdered mycelium of a Trichoderma harzianum strain by extraction with guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion (Chirgwin et al., (1979), Biochemistry, 18, 5294- 5299). Poly(A)+RNA was isolated by oligo(dT)cellulose affinity chromatography (Aviv and Leder, (1972) , Proc. Natl. Acad. Sci. U.S.A., 69, 1408-1412). Double-stranded cDNA was synthesized from 5 μg of poly(A)+RNA as described (Gubler and Hoffman, (1983), Gene, 25, 263-269; Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, Cold Spring Harbot Laboratory) except that 25 ng of random hexanucleotide primers (Gibco BRL, USA) were included in the first strand synthesis. A cDNA library consisting of 1.5 x 10⁶ clones was constructed in the yeast expression vector pYES 2.0 as described previously (Kofod et al., (1994), J. Biol. Chem., 261, 8407-8413) . Plasmid DNA from a cDNA library pool was transformed into S. cerevisiae W3124 (van den Hazel et al., (1992), Eur. J. Biochem. , 207, 277-283) by electroporation (Becker and Guarente, (1991), Methods Enzymol., 194, 182-187) and the transformants were plated on SC agar (Sherman, (1991) , Methods Enzymol. 194, 3-21) containing 2% glucose. After incubation at 30°C for 3 to 4 days, the colonies were replicated onto SC agar plates containing 2% galactose and incubated for 3 days at 30°C before the yeast colonies were overlayered with 1% agarose containing 0.1 M citric acid/sodium citrate buffer pH 5.0 and 0.001% 4-methylumbelliferyl N- acetylglucosamine (MUF-GlcNAc) .

Exochitinase positive clones were identified under UV-light by the formation of fluorescent halos. Total DNA from the positive yeast colonies was isolated and the insert containing pYES 2.0 clones were rescued by transformation of E. coli MC 1061 (Meissner et al., (1987), Proc. Natl. Acad. Sci. U.S.A., 84, 4171-4176) to ampicillin resistance.

Nucleotide sequence analysis The nucleotide sequence of the cDNA insert was determined from both strands by the dideoxy chain termination method (Sanger et al., (1977), Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467) using Qiagen purified plasmid DNA, the Sequenase®kit or synthetic oligonucleotide primers. Analysis of the sequence date were performed according to Devereux et al., (1984), Nucleic Acids Res., 12, 387-395). The full length cDNA sequences are shown in SEQ ID no. 1 and SEQ ID No. 3, respectively, and the corresponding amino acid sequences in SEQ ID No. 2 and SEQ ID no. 4, respectively.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novo Nordis A/S

(B) STREET: Novo Alle

(C) CITY: Bagsvaerd (E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP): DK-2800

(G) TELEPHONE: +45 4444 8888 (H) TELEFAX: +45 4449 3256 (ii) TITLE OF INVENTION: An enzyme with exochitinase activity

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30B (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2000 base pairs (B) TYPE: nucleic acid

(C) STRANDΞDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(B) STRAIN: Trichoderma harzianum CBS 243.71

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION:86..1819

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GACATCTCCA CCATAGAGTC GACTCATTGC TGGCATACGG AGCATTCCAA TCTTACTCGT 60

AGTAGTGTTA TTGCCATCGC TCATC ATG CTG CCC AAG GCG ATC ATC GCG ATT 112

Met Leu Pro Lys Ala He He Ala He 1 5

GCC GCA TTG GCT TTC AGC CCA GCA AAT GCG CTG TGG CCC ATT CCT CAG 160 Ala Ala Leu Ala Phe Ser Pro Ala Asn Ala Leu Trp Pro He Pro Gin 10 15 20 25

AAG ATC TCG ACC GGA GAC AGC GTG CTC TTT ATT GAC CAG GCT GTT AGG 208 Lys He Ser Thr Gly Asp Ser Val Leu Phe He Asp Gin Ala Val Arg 30 35 40 GTG ACT TAC AAT GGA GTA CCG ATC ATC CCT ATC GGC TAC AAC CCA CCG 256 Val Thr Tyr Asn Gly Val Pro He He Pro He Gly Tyr Asn Pro Pro 45 50 55

GCC AGC TCC AAC TTC GAC AGC AGG CAA ATC GTC CAG GCG GCT GTC TCG 304 Ala Ser Ser Asn Phe Asp Ser Arg Gin He Val Gin Ala Ala Val Ser 60 65 70

CGC GCT TTC CAA AAC ATC TTC AGC ACC AAC TAT GTG CCA TGG AAG CTT 352 Arg Ala Phe Gin Asn He Phe Ser Thr Asn Tyr Val Pro Trp Lys Leu 75 80 85

CAC CCG CGT AAC AGC AAC TTT GAG CCG AAG GTG GCC CCT CAG AAC CGA 400

His Pro Arg Asn Ser Asn Phe Glu Pro Lys Val Ala Pro Gin Asn Arg

90 95 100 105

ATC CAG TCC ATC TCA ATT CAG CAG ACT GGA AAG GAT ACG TCC AAG ACG 448 He Gin Ser He Ser He Gin Gin Thr Gly Lys Asp Thr Ser Lys Thr 110 115 120

TTC AAG CCG CGC GCC GGA GAC GTT GAT GAG TCG TAC TCT TTG ACC ATT 496 Phe Lys Pro Arg Ala Gly Asp Val Asp Glu Ser Tyr Ser Leu Thr He 125 130 135

TCC AAG AAT GGA CAG GTC AAC ATC AGT GCC AAG TCT TCT ACT GGT GTG 544 Ser Lys Asn Gly Gin Val Asn He Ser Ala Lys Ser Ser Thr Gly Val 140 145 150 CTG CAC GCC CTC GAG ACC TTC TCG CAG CTT TTC TAC AAG CAC TCT GCT 592 Leu His Ala Leu Glu Thr Phe Ser Gin Leu Phe Tyr Lys His Ser Ala 155 160 165

GGA CCT TTC TAC TAT ACG ACT CAG GCT CCC GTG TCC ATC ACA GAC GCT 640 Gly Pro Phe Tyr Tyr Thr Thr Gin Ala Pro Val Ser He Thr Asp Ala 170 175 180 185

CCC AAA TAT CCC CAC CGT GGC ATC ATG CTT GAC CTT GCC CGT AAC TAT 688 Pro Lys Tyr Pro His Arg Gly He Met Leu Asp Leu Ala Arg Asn Tyr 190 195 200

CAA ACC ATT GAT GAC ATC AAG AGG ACC ATT GAC GCC ATG TCG TGG AAC 736 Gin Thr He Asp Asp He Lys Arg Thr He Asp Ala Met Ser Trp Asn 205 210 215

AAG CTT AAC CGC CTG CAC TTG CAC ATC ACC GAC TCT CAG TCG TGG CCG 784 Lys Leu Asn Arg Leu His Leu His He Thr Asp Ser Gin Ser Trp Pro 220 225 230 CTG GTG ATC CCC TCG CTG CCT AAG CTG TCC CAG GCC GGT GCC TAC CAC 832 Leu Val He Pro Ser Leu Pro Lys Leu Ser Gin Ala Gly Ala Tyr His 235 240 245

CCC AGC CTC GTC TAC ACT CCC GCA GAC CTT GCT GGC ATT TTC CAG TAC * 880 Pro Ser Leu Val Tyr Thr Pro Ala Asp Leu Ala Gly He Phe Gin Tyr 250 255 260 265

GGT GTC GCC CGC GGT GTT GAG GTC ATT ACG GAG ATC GAT ATG CCT GGC 928 Gly Val Ala Arg Gly Val Glu Val He Thr Glu He Asp Met Pro Gly 270 275 280

CAC ATC GGT GTT ATC GAG CTC GCT TAC AGC GAT CTC ATT GTT GCC TAC 976 His He Gly Val He Glu Leu Ala Tyr Ser Asp Leu He Val Ala Tyr 285 290 295

GAA GAG ATG CCT TAC CAG TAC TAC TGC GCC GAG CCA CCT TGC GGT GCC 1024 Glu Glu Met Pro Tyr Gin Tyr Tyr Cys Ala Glu Pro Pro Cys Gly Ala 300 ^' 305 310 TTT TCC ATC AAC AAC ACC AAG GTG TAC AGC TTC CTC GAT ACC CTG TTC 1072

Phe Ser He Asn Asn Thr Lys Val Tyr Ser Phe Leu Asp Thr Leu Phe 315 320 325

GAC GAC CTT TTG CCT CGC GTC GCT CCT TAC AGC GCG TAC TTC CAC ACC 1120 Asp Asp Leu Leu Pro Arg Val Ala Pro Tyr Ser Ala Tyr Phe His Thr 330 335 340 345

GGT GGT GAC GAG CTC AAC GCT AAC GAC TCC ATG CTC GAC TCT CAC ATC 1168 Gly Gly Asp Glu Leu Asn Ala Asn Asp Ser Met Leu Asp Ser His He 350 355 360

AAG AGC AAC GAG ACC TCC GTT CTG CAA CCT CTG CTG CAA AAG TTC ATC 1216 Lys Ser Asn Glu Thr Ser Val Leu Gin Pro Leu Leu Gin Lys Phe He 365 370 375

AAC TTT GCC CAC TCC AAG GTC CGT GCC GCG GGC TTG TCG CCA TTT GTC 1264 Asn Phe Ala His Ser Lys Val Arg Ala Ala Gly Leu Ser Pro Phe Val 380 385 390

TGG GAG GAG ATG GTC ACC ACC TGG AAC CTG ACC CTC GGC AGC GAC ACC 1312 Trp Glu Glu Met Val Thr Thr Trp Asn Leu Thr Leu Gly Ser Asp Thr 395 400 405

GTC GTT CAG TCG TGG CTG GGT GGC GAT GCC GTC AAG AAC CTG GCT GAG 1360 Val Val Gin Ser Trp Leu Gly Gly Asp Ala Val Lys Asn Leu Ala Glu 410 415 420 425 AGC GGC CAC AAG GTC ATT GAC ACC GAC TAC AAC TTC TAC TAC TTG GAC 1408 Ser Gly His Lys Val He Asp Thr Asp Tyr Asn Phe Tyr Tyr Leu Asp 430 435 440

TGC GGC CGT GGT CAA TGG GTC AAC TTC CCT CCA GGA GAC TCC TAC AAC 1456 Cys Gly Arg Gly Gin Trp Val Asn Phe Pro Pro Gly Asp Ser Tyr Asn 445 450 455

ACC TAC TAC CCA TTC AAC GAC TGG TGC CAG CCC ACC AAG AAC TGG AGG 1504 Thr Tyr Tyr Pro Phe Asn Asp Trp Cys Gin Pro Thr Lys Asn Trp Arg 460 465 470

CTC ATC TAC TCT CAC GAC CCT GCA GCC AAC GTG TCT GCT TCG GCT GCC 1552 Leu He Tyr Ser His Asp Pro Ala Ala Asn Val Ser Ala Ser Ala Ala 475 480 485

AAG AAC GTC CTG GGA GGA GAG CTT GCC ATT TGG AGC GAG ATG ATT GAC 1600 Lys Asn Val Leu Gly Gly Glu Leu Ala He Trp Ser Glu Met He Asp 490 495 500 505 GCC AGC AAC CTG GAC AAC ATC ATC TGG CCT CGT GGC AGC GCC GCC GGT 1648 Ala Ser Asn Leu Asp Asn He He Trp Pro Arg Gly Ser Ala Ala Gly 510 515 520

GAG GTT TGG TGG TCC GGC AAT ACC GAT GCC TCT GGT GAG CAG CGC AGC 1696 Glu Val Trp Trp Ser Gly Asn Thr Asp Ala Ser Gly Glu Gin Arg Ser 525 530 535

CAG CTG GAC GTT GTT CCT CGT CTG AAC GAG TTC CGA GAA CGC TTG CTT 1744 Gin Leu Asp Val Val Pro Arg Leu Asn Glu Phe Arg Glu Arg Leu Leu 540 545 550

GCT CGT GGT GTC AGC GCG TTC CCC ATC CAG ATG ACC TAC TGC ACT CAG 1792 Ala Arg Gly Val Ser Ala Phe Pro He Gin Met Thr Tyr Cys Thr Gin 555 560 565

CTC AAC GCC ACT GCC TGC ACA CTA TTT TAAGTCTAAG ATGACTTTTT 1839

Leu Asn Ala Thr Ala Cys Thr Leu Phe 570 575 CTTTTATTGG GCAGGGTTTT TTCTATTTTT CACGTATTAT CATTAGTGTA CAGTGATTAA 1899

AACAGGTATG GCTTAAGAGG AGCTGGGAGG GTATCCGGCT TGGGGCGGTA TATTATTAAC 1959

TGTATATAAT TCAAATTCAT CTACATATAT GTTATGAAAA A 2000

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 578 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Leu Pro Lys Ala He He Ala He Ala Ala Leu Ala Phe Ser Pro 1 5 10 15

Ala Asn Ala Leu Trp Pro He Pro Gin Lys He Ser Thr Gly Asp Ser 20 25 30 Val Leu Phe He Asp Gin Ala Val Arg Val Thr Tyr Asn Gly Val Pro 35 40 45

He He Pro He Gly Tyr Asn Pro Pro Ala Ser Ser Asn Phe Asp Ser 50 55 60

Arg Gin He Val Gin Ala Ala Val Ser Arg Ala Phe Gin Asn He Phe 65 70 75 80

Ser Thr Asn Tyr Val Pro Trp Lys Leu His Pro Arg Asn Ser Asn Phe 85 90 95

Glu Pro Lys Val Ala Pro Gin Asn Arg He Gin Ser He Ser He Gin 100 105 110 Gin Thr Gly Lys Asp Thr Ser Lys Thr Phe Lys Pro Arg Ala Gly Asp 115 120 125

Val Asp Glu Ser Tyr Ser Leu Thr He Ser Lys Asn Gly Gin Val Asn 130 135 140

He Ser Ala Lys Ser Ser Thr Gly Val Leu His Ala Leu Glu Thr Phe 145 150 155 160

Ser Gin Leu Phe Tyr Lys His Ser Ala Gly Pro Phe Tyr Tyr Thr Thr 165 170 175

Gin Ala Pro Val Ser He Thr Asp Ala Pro Lys Tyr Pro His Arg Gly 180 185 190 He Met Leu Asp Leu Ala Arg Asn Tyr Gin Thr He Asp Asp He Lys 195 200 205

Arg Thr He Asp Ala Met Ser Trp Asn Lys Leu Asn Arg Leu His Leu 210 215 220

His He Thr Asp Ser Gin Ser Trp Pro Leu Val He Pro Ser Leu Pro 225 230 235 240

Lys Leu Ser Gin Ala Gly Ala Tyr His Pro Ser Leu Val Tyr Thr Pro 245 250 255

Ala Asp Leu Ala Gly He Phe Gin Tyr Gly Val Ala Arg Gly Val Glu 260 265 270 Val He Thr Glu He Asp Met Pro Gly His He Gly Val He Glu Leu 275 280 285

Ala Tyr Ser Asp Leu He Val Ala Tyr Glu Glu Met Pro Tyr Gin Tyr 290 295 300

Tyr Cys Ala Glu Pro Pro Cys Gly Ala Phe Ser He Asn Asn Thr Lys 305 310 315 320 Val Tyr Ser Phe Leu Asp Thr Leu Phe Asp Asp Leu Leu Pro Arg Val 325 330 335

Ala Pro Tyr Ser Ala Tyr Phe His Thr Gly Gly Asp Glu Leu Asn Ala 340 345 350

Asn Asp Ser Met Leu Asp Ser His He Lys Ser Asn Glu Thr Ser Val 355 360 365 Leu Gin Pro Leu Leu Gin Lys Phe He Asn Phe Ala His Ser Lys Val 370 375 380

Arg Ala Ala Gly Leu Ser Pro Phe Val Trp Glu Glu Met Val Thr Thr 385 390 395 400

Trp Asn Leu Thr Leu Gly Ser Asp Thr Val Val Gin Ser Trp Leu Gly 405 410 415

Gly Asp Ala Val Lys Asn Leu Ala Glu Ser Gly His Lys Val He Asp 420 425 430

Thr Asp Tyr Asn Phe Tyr Tyr Leu Asp Cys Gly Arg Gly Gin Trp Val 435 440 445 Asn Phe Pro Pro Gly Asp Ser Tyr Asn Thr Tyr Tyr Pro Phe Asn Asp 450 455 460

Trp Cys Gin Pro Thr Lys Asn Trp Arg Leu He Tyr Ser His Asp Pro

465 470 475 480

Ala Ala Asn Val Ser Ala Ser Ala Ala Lys Asn Val Leu Gly Gly Glu

485 490 495

Leu Ala He Trp Ser Glu Met He Asp Ala Ser Asn Leu Asp Asn He 500 505 510

He Trp Pro Arg Gly Ser Ala Ala Gly Glu Val Trp Trp Ser Gly Asn 515 520 525 Thr Asp Ala Ser Gly Glu Gin Arg Ser Gin Leu Asp Val Val Pro Arg 530 535 540

Leu Asn Glu Phe Arg Glu Arg Leu Leu Ala Arg Gly Val Ser Ala Phe 545 550 555 560

Pro He Gin Met Thr Tyr Cys Thr Gin Leu Asn Ala Thr Ala Cys Thr 565 570 575

Leu Phe

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2239 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(B) STRAIN: Trichoderma harzianum CBS 243.71

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION:282..2086

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CTGAGAAGCG GCACTTGCTG ATCTGCGTGG AACTTGGGGT TACAACGCAC CGGATAGCTC 60

ATCTCCCCAG GACCCCGGAA CTGGAGCTGG AACTGGAATT GGAGCTGGAG CGGACCCAGG 120 CCGGAGACGA GAAACACAGT GAATCACTCC TGCAAGGGGC GGGACAGGAA CGTGGACAGT 180

ATTTAGTTTA AGCAGCTGTC CCAGAGCTGT TCGCCCTGCT TCCAAGCTCG TGTGGCCTGA 240

CCCTGTATAA ACTCATTACG ACCATCAGCT CACAGCCGAC A ATG TTT TCC AGG 293 Met Phe Ser Arg

1

GCC ATT GTC GCC GCA TTG GCC CTG AGC GGC CCG GCT TTT GCC CTG TGG 341 Ala He Val Ala Ala Leu Ala Leu Ser Gly Pro Ala Phe Ala Leu Trp 5 10 15 20

CCC GTG CCT AAA CAC TCC TCG ACC GGC AAT GAC ACG CTC TTT ATT GAC 389 Pro Val Pro Lys His Ser Ser Thr Gly Asn Asp Thr Leu Phe He Asp 25 30 35

CAG ACG GTC CAG GTT ACC TAC AAT GGT GAA CAG GTG TGG TGG ACT CCT 437 Gin Thr Val Gin Val Thr Tyr Asn Gly Glu Gin Val Trp Trp Thr Pro 40 45 50 CCA TAT GAT GAC CCC GGA TCC CCG GAC TTT GCT GAG ACC AGG ATC GAT 485 Pro Tyr Asp Asp Pro Gly Ser Pro Asp Phe Ala Glu Thr Arg He Asp 55 60 65

GAC CAA CAG GTT ACT TAC ACG GCC GGC TAC GTG CCT CCC AGC GGA CCG 533 Asp Gin Gin Val Thr Tyr Thr Ala Gly Tyr Val Pro Pro Ser Gly Pro 70 75 80

CAT TTC ACC AGC AAG GAA ATC GTT CAA GGC GGC GTC TCG CGG ACA TTC 581 His Phe Thr Ser Lys Glu He Val Gin Gly Gly Val Ser Arg Thr Phe 85 90 95 100

GGC GCC ATC TTC CAG CAG GGC TTT GTG CCG TGG ATG CTG CGT GAA CGT 629 Gly Ala He Phe Gin Gin Gly Phe Val Pro Trp Met Leu Arg Glu Arg 105 110 115

GAT TCG AAC TCT GAA CCG AAT CTA GGC GGA ACG CGG ATC CGG ACA CTG 677 Asp Ser Asn Ser Glu Pro Asn Leu Gly Gly Thr Arg He Arg Thr Leu 120 125 130 CAG ATT ATA CAG ACT CAG CAC GAT TCT GCG AAT ACC TTC AAG CCT CTG 725 Gin He He Gin Thr Gin His Asp Ser Ala Asn Thr Phe Lys Pro Leu 135 140 145 AAT GGC GCA GTG AAT GAA TCC TAT GCC CTG GAT GTC GAC GCA AAG GGC 773 Asn Gly Ala Val Asn Glu Ser Tyr Ala Leu Asp Val Asp Ala Lys Gly 150 155 160 CAC GCA TCT CTC ACC GCT CCG TCG TCA ACG GGC ATC CTT CGA GGC CTT 821 His Ala Ser Leu Thr Ala Pro Ser Ser Thr Gly He Leu Arg Gly Leu 165 170 175 180

GAG ACC TTC TCC CAG CTC TTC TTC AAG CAT AGC TCC GGC ACT GCT TGG 869 Glu Thr Phe Ser Gin Leu Phe Phe Lys His Ser Ser Gly Thr Ala Trp

185 190 195

TAT ACG CAG CTT GCA CCT GTT TCG ATC CGC GAT GAG CCC AAG TAT CCT 917 Tyr Thr Gin Leu Ala Pro Val Ser He Arg Asp Glu Pro Lys Tyr Pro 200 205 210

CAC CGC GGC CTC CTG TTG GAT GTC AGC CGC CAT TGG TTC GAG GTT TCC 965 His Arg Gly Leu Leu Leu Asp Val Ser Arg His Trp Phe Glu Val Ser 215 220 225

GAC ATT GAG CGC ACT ATC GAT GCT CTG GCC ATG AAC AAA ATG AAT GTG 1013 Asp He Glu Arg Thr He Asp Ala Leu Ala Met Asn Lys Met Asn Val 230 235 240 CTG CAT CTG CAC GCT ACT GAC ACG CAG TCA TGG CCG CTG GAG ATT CCA 1061 Leu His Leu His Ala Thr Asp Thr Gin Ser Trp Pro Leu Glu He Pro 245 250 255 260

TCC CTG CCT CTG CTG GCT GAG AAG GGC GCC TAT CAC AAG GGT TTG AGC 1109 Ser Leu Pro Leu Leu Ala Glu Lys Gly Ala Tyr His Lys Gly Leu Ser

265 270 275

TAC TCG CCA AGC GAT CTT GCG AGC ATC CAA GAA TAT GGT GTT CAT CGA 1157 Tyr Ser Pro Ser Asp Leu Ala Ser He Gin Glu Tyr Gly Val His Arg 280 285 290

GGT GTC CAG GTC ATT GTA GAG ATT GAT ATG CCG GGC CAC GTT GGA ATC 1205 Gly Val Gin Val He Val Glu He Asp Met Pro Gly His Val Gly He 295 300 305

GAC AAG GCA TAC CCC GGG CTT AGC AAC GCC TAC GGA GTC AAC CCG TGG 1253 Asp Lys Ala Tyr Pro Gly Leu Ser Asn Ala Tyr Gly Val Asn Pro Trp 310 315 320 CAG TGG TAC TGC GCC CAG CCG CCC TGC GGA TCT TTC AAG CTG AAC AAC 1301 Gin Trp Tyr Cys Ala Gin Pro Pro Cys Gly Ser Phe Lys Leu Asn Asn 325 330 335 340

ACG GAT GTC GAA AAG TTC ATT GAC AAG CTG TTT GAA GAT TTG CTG CCG 1349 Thr Asp Val Glu Lys Phe He Asp Lys Leu Phe Glu Asp Leu Leu Pro

345 350 355

CGT CTT TCG CCG TAC TCG GCC TAC TTT CAC ACT GGT GGC GAT GAG TAC 1397 Arg Leu Ser Pro Tyr Ser Ala Tyr Phe His Thr Gly Gly Asp Glu Tyr 360 365 370

AAG GCG AAC AAC TCG CTG CTC GAC CCG GCC CTT CGC ACA AAC GAC ATG 1445 Lys Ala Asn Asn Ser Leu Leu Asp Pro Ala Leu Arg Thr Asn Asp Met 375 380 385

AAC ACC CTG CAG CCG ATG CTG CAG CGC TTC TTG GAC CAC GTG CAT GGC 1493 Asn Thr Leu Gin Pro Met Leu Gin Arg Phe Leu Asp His Val His Gly 390 395 400 AAA GTT CGT GAT CTG GGA CTC GTT CCC ATG GTT TGG GAA GAA ATG ATT 1541 Lys Val Arg Asp Leu Gly Leu Val Pro Met Val Trp Glu Glu Met He 405 410 415 420 CTG GAT TGG AAC GCA ACT CTG GGC AAG GAT GTC GTT GCT CAA ACG TGG 1589 Leu Asp Trp Asn Ala Thr Leu Gly Lys Asp Val Val Ala Gin Thr Trp 425 430 435 CTT GGC GGA GGA GCG ATT CAG AAG CTT GCT CAG GCT GGA TAC AAG GTT 1637 Leu Gly Gly Gly Ala He Gin Lys Leu Ala Gin Ala Gly Tyr Lys Val 440 445 450

ATT GAC AGC AGC AAT GAC TTT TAC TAT CTC GAC TGT GGT CGT GGT GAG 1685 He Asp Ser Ser Asn Asp Phe Tyr Tyr Leu Asp Cys Gly Arg Gly Glu 455 460 465

TGG CTC GAT TTT GCC AAT GGT GAC CCC TTT AAC AAC AAC TAT CCC TTT 1733 Trp Leu Asp Phe Ala Asn Gly Asp Pro Phe Asn Asn Asn Tyr Pro Phe 470 475 480

CTC GAC TGG TGC GAC CCG ACC AAA AAC TGG AAG CTC ATG TAC TCA CAC 1781 Leu Asp Trp Cys Asp Pro Thr Lys Asn Trp Lys Leu Met Tyr Ser His 485 490 495 500

GAG CCC ACG GAC GGC GTG TCC GAT GAT CTC AAG AAG AAT GTC ATT GGA 1829 Glu Pro Thr Asp Gly Val Ser Asp Asp Leu Lys Lys Asn Val He Gly 505 510 515 GGC GAA GTT GCT GTC TGG ACT GAG ACC ATC GAT CCG ACC AGC TTG GAC 1877 Gly Glu Val Ala Val Trp Thr Glu Thr He Asp Pro Thr Ser Leu Asp 520 525 530

TCC ATC ATC TGG CCG CGA GCG GGA GCG GCC GCT GAG ATT TGG TGG TCG 1925 Ser He He Trp Pro Arg Ala Gly Ala Ala Ala Glu He Trp Trp Ser 535 540 545

GGC AAG ATC GAT GAG AAG GGC CAG AAC CGA TCA CAG ATT GAT GCA CGG 1973 Gly Lys He Asp Glu Lys Gly Gin Asn Arg Ser Gin He Asp Ala Arg 550 555 560

CCA AGA TTA TCG GAG CAG CGA GAG CGC ATG TTG GCG AGG GGA GTT CGA 2021 Pro Arg Leu Ser Glu Gin Arg Glu Arg Met Leu Ala Arg Gly Val Arg 565 570 575 580

GGA ACG CCG ATT ACG CAG CTG TGG TGT AGT CAG GTT GAT GTT CAT AAC 2069 Gly Thr Pro He Thr Gin Leu Trp Cys Ser Gin Val Asp Val His Asn 585 590 595 TGC GAG TCT GGG AAT TA CTGATGCGGG TTGATGAACA AAGTATGTAA 2116

Cys Glu Ser Gly Asn 600

TGTGGTATAT ATGAATGTTT CTTTTTCACG CTGCTGTTAA AGGCCGGGGA CGTCTCGTTT 2176

GTGATGACGG TTAGACTGAA AATCACTTAT AATGAATTCA AGTCATTCAA GATGAAAAAA 2236

AAA 2239

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 601 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

( ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :

Met Phe Ser Arg Ala He Val Ala Ala Leu Ala Leu Ser Gly Pro Ala 1 5 10 15 Phe Ala Leu Trp Pro Val Pro Lys His Ser Ser Thr Gly Asn Asp Thr 20 25 30

Leu Phe He Asp Gin Thr Val Gin Val Thr Tyr Asn Gly Glu Gin Val 35 40 45

Trp Trp Thr Pro Pro Tyr Asp Asp Pro Gly Ser Pro Asp Phe Ala Glu 50 55 60

Thr Arg He Asp Asp Gin Gin Val Thr Tyr Thr Ala Gly Tyr Val Pro 65 70 75 80

Pro Ser Gly Pro His Phe Thr Ser Lys Glu He Val Gin Gly Gly Val 85 90 95

Ser Arg Thr Phe Gly Ala He Phe Gin Gin Gly Phe Val Pro Trp Met 100 105 110

Leu Arg Glu Arg Asp Ser Asn Ser Glu Pro Asn Leu Gly Gly Thr Arg 115 120 125

He Arg Thr Leu Gin He He Gin Thr Gin His Asp Ser Ala Asn Thr 130 135 140 Phe Lys Pro Leu Asn Gly Ala Val Asn Glu Ser Tyr Ala Leu Asp Val 145 150 155 160

Asp Ala Lys Gly His Ala Ser Leu Thr Ala Pro Ser Ser Thr Gly He 165 170 175

Leu Arg Gly Leu Glu Thr Phe Ser Gin Leu Phe Phe Lys His Ser Ser 180 185 190

Gly Thr Ala Trp Tyr Thr Gin Leu Ala Pro Val Ser He Arg Asp Glu 195 200 205

Pro Lys Tyr Pro His Arg Gly Leu Leu Leu Asp Val Ser Arg His Trp 210 215 220 Phe Glu Val Ser Asp He Glu Arg Thr He Asp Ala Leu Ala Met Asn 225 230 235 240

Lys Met Asn Val Leu His Leu His Ala Thr Asp Thr Gin Ser Trp Pro 245 250 255

Leu Glu He Pro Ser Leu Pro Leu Leu Ala Glu Lys Gly Ala Tyr His 260 265 270

Lys Gly Leu Ser Tyr Ser Pro Ser Asp Leu Ala Ser He Gin Glu Tyr 275 280 285

Gly Val His Arg Gly Val Gin Val He Val Glu He Asp Met Pro Gly 290 295 300 His Val Gly He Asp Lys Ala Tyr Pro Gly Leu Ser Asn Ala Tyr Gly 305 310 315 320

Val Asn Pro Trp Gin Trp Tyr Cys Ala Gin Pro Pro Cys Gly Ser Phe 325 330 335

Lys Leu Asn Asn Thr Asp Val Glu Lys Phe He Asp Lys Leu Phe Glu 340 345 350

Asp Leu Leu Pro Arg Leu Ser Pro Tyr Ser Ala Tyr Phe His Thr Gly 355 360 365

Gly Asp Glu Tyr Lys Ala Asn Asn Ser Leu Leu Asp Pro Ala Leu Arg 370 375 380 Thr Asn Asp Met Asn Thr Leu Gin Pro Met Leu Gin Arg Phe Leu Asp

385 390 395 400

His Val His Gly Lys Val Arg Asp Leu Gly Leu Val Pro Met Val Trp 405 410 415

Glu Glu Met He Leu Asp Trp Asn Ala Thr Leu Gly Lys Asp Val Val 420 425 430

Ala Gin Thr Trp Leu Gly Gly Gly Ala He Gin Lys Leu Ala Gin Ala 435 440 445

Gly Tyr Lys Val He Asp Ser Ser Asn Asp Phe Tyr Tyr Leu Asp Cys 450 455 460

Gly Arg Gly Glu Trp Leu Asp Phe Ala Asn Gly Asp Pro Phe Asn Asn 465 470 475 480 Asn Tyr Pro Phe Leu Asp Trp Cys Asp Pro Thr Lys Asn Trp Lys Leu

485 490 495

Met Tyr Ser His Glu Pro Thr Asp Gly Val Ser Asp Asp Leu Lys Lys 500 505 510

Asn Val He Gly Gly Glu Val Ala Val Trp Thr Glu Thr He Asp Pro 515 520 525

Thr Ser Leu Asp Ser He He Trp Pro Arg Ala Gly Ala Ala Ala Glu 530 535 540

He Trp Trp Ser Gly Lys He Asp Glu Lys Gly Gin Asn Arg Ser Gin 545 550 555 560 He Asp Ala Arg Pro Arg Leu Ser Glu Gin Arg Glu Arg Met Leu Ala

565 570 575

Arg Gly Val Arg Gly Thr Pro He Thr Gin Leu Trp Cys Ser Gin Val 580 585 590

Asp Val His Asn Cys Glu Ser Gly Asn 595 600

Claims

1. A DNA construct comprising a DNA sequence encoding an enzyme exhibiting exochitinase activity, which DNA sequence a) comprises the DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3, or b) comprises an analogue of the DNA sequence shown in SEQ ID no. 1 or SEQ ID no 3, which i) is homologous with the DNA sequences shown in SEQ ID no. 1 or SEQ ID no. 3, and/or ii) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID no. 1 or SEQ ID no. 3, and/or iii) encodes a polypeptide which is homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID No. 1 or SEQ ID no. 3, and/or iv) encodes a polypeptide which is immunologically reactive with an antibody raised against a purified exochitinase shown in SEQ ID no. 2 derived from

Saccharomyces cerevisiae DSM no. 9944 or against a purified exochitinase shown in SEQ ID no. 4 derived from Saccharomyces cerevisiae DSM no. 9945.

2. A DNA construct comprising a DNA sequence encoding an enzyme exhibiting exochitinase activity, which DNA sequence comprises at least a partial sequence of the sequence shown in SEQ ID No. 1 or SEQ ID no. 3.

3. The DNA construct according to claim 1 or 2, in which the DNA sequence encoding an enzyme exhibiting exochitinase activity is obtainable from a microorganism.

4. The DNA construct according to claim 3, in which the DNA sequence is obtainable from a filamentous fungus or a yeast.

5. The DNA construct according to claim 4, in which is the DNA sequence is obtainable from a strain of Saccharomyces, Aspergillus, Trichoderma, Penicillium, Fuεarium, Gliocladium, Aphanocladium, or Humicola, or a mutant thereof.

6. The DNA construct according to claim 5, in which the DNA sequence is obtainable from a strain of Trichoderma in par¬ ticular a strain of T. harzianum , or T. reesei , or T. viride, T. longibrachiatum, or T. koningii , or a mutant thereof.

7. The DNA construct according to claim 5, in which the DNA sequence is obtainable from a strain of Aspergillus, in particular a strain of A. aculeatus or A. niger, or a mutant thereof.

8. The DNA construct according to claim 5, in which the DNA sequence is obtainable from a strain of Fusarium, in particular a strain of F. oxysporum, or a mutant thereof.

9. The DNA construct according to claim 5, in which the DNA sequence is obtainable from a strain of Gliocladium, in particular Gliocladium virens, or a mutant thereof.

10. The DNA construct according to claim 5, in which the DNA sequence is obtainable from a strain of Aphanocladium, in particular Aphanocladium album, or a mutant thereof.

11. The DNA construct according to claim 5, in which the DNA sequence is obtainable from a strain of Humicola, or a mutant thereof.

12. The DNA construct according to claim 5, in which the DNA sequence is obtainable from a strain of Saccharomyces , in particular Saccharomyces cerevisiae, or a mutant thereof.

13. The DNA construct according to claim 12, in which the DNA sequence is isolated from Saccharomyces cerevisiae DSM No. 9944 or DSM no. 9945.

14. A recombinant expression vector comprising a DNA construct according to any of claims 1-13.

15. A cell comprising a DNA construct according to any of claims 1-13 or a recombinant expression vector according to claim 14.

16. A cell according to claim 15, which is an eukaryotic cell, in particular a fungal cell, such as a yeast cell or a filamentous fungal cell.

17. A cell according to claim 16, wherein the cell belongs to a strain of Trichodrma , in particular a strain of Trichoderma harzianum or Trichoderma reesie.

18. A method of producing an enzyme exhibiting exochitinase activity, the method comprising culturing a cell according to any of claims 15-17 under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.

19. An enzyme exhibiting exochitinase activity, which enzyme a) is encoded by a DNA construct according to any of claims 1- 13, and/or b) produced by the method according to claim 18, and/or c) is immunologically reactive with an antibody raised against a purified exochitinase derived from Saccharomyces cerevisiae, DSM no. 9944 shown in SEQ ID no. 1 or DSM no. 9945 shown in SEQ ID no. 3.

20. An enzyme preparation useful for modification or degrada¬ tion of fungal, crustacean, or nematode cell wall components, said preparation being enriched in an enzyme exhibiting exochitinase activity according to claim 19.

21. A preparation according to claim 20, which additionally comprises an enzyme with chitinolytic, proteolytic, β- glucanolytic, or mannanolytic activity.

22. Use of an enzyme according to claim 19 or an enzyme prepa¬ ration according to claim 21 or 22 for modification or degra¬ dation of fungal, crustacean, or nematode cell wall components.

23. The use according to claim 22 for plant protection purposes.

24. The use according to claim 23 for coating seeds.

25. The use according to claim 23 for spraying plants.

26. The use according to claim 22 for pharmaceutical purposes.

27. The use according to claim 26 for wound dressing.

28. Use of a DNA construct according to claims 1 to 13 or an expression vector according to claim 14 for producing transgenic plants.

29. An isolated substantially pure culture of Saccharomyces cerevisiae DSM no. 9944.

30. An isolated substantially pure culture of Saccharomyces cerevisiae DSM no. 9945