WO1998036056A1

WO1998036056A1 - An enzyme with endo-1,3(4)-beta-glucanase activity

Info

Publication number: WO1998036056A1
Application number: PCT/DK1998/000057
Authority: WO
Inventors: Marie-Louise Bang; Thomas Sandal
Original assignee: Novo Nordisk A/S
Priority date: 1997-02-14
Filing date: 1998-02-13
Publication date: 1998-08-20
Also published as: AU5853098A

Abstract

The present invention relates to an enzyme with beta-glucanase activity, a cloned DNA sequence encoding the enzyme with beta-glucanase activity, a method of producing the enzyme, an enzyme composition comprising said enzyme with beta-glucanase activity, and the use of said enzyme and enzyme composition for a number of industrial applications. The enzyme may be obtained from Phaffia rhodozyma.

Description

AN ENZYME WITH ENDO-l,3(4)-BETA-GLUCANASE ACTIVITY

FIELD OF INVENTION

The present invention relates to an enzyme with beta-glucanase activity, a cloned DNA sequence encoding the enzyme with beta- glucanase activity, a method of producing the enzyme, an enzyme composition comprising said enzyme with beta-glucanase activity, and the use of said enzyme and enzyme composition for a number of industrial applications.

SUMMARY OF THE INVENTION

The present inventors have found that an enzyme exhibiting beta-glucanase activity may be obtained from a strain of the genus Phaffia , more specifically Phaffia rhodozyma, and have succeeded in cloning a DNA sequence encoding said enzyme.

Accordingly, in a first aspect the invention relates to a cloned DNA sequence encoding an enzyme exhibiting beta- glucanase activity, which DNA sequence is selected from the group comprising of:

(a) the beta-glucanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in Escherichia coli DSM 11342; (b) the DNA sequence shown in positions 1-1275 in SEQ ID NO 1 or its complementary strand;

(c) an analogue of the DNA sequence defined in (a) or (b) which is at least 70% homologous with said DNA sequence;

(d) a DNA sequence which hybridizes with the DNA sequence shown in positions 1-1275 in SEQ ID NO 1 at low stringency;

(e) a DNA sequence which, because of the degeneracy of the genetic code, does not hybridize with the sequences of (b) or (d) , but which codes for a polypeptide having exactly the same amino acid sequences as the polypeptide encoded by these DNA sequences; and

(f) a DNA sequence which is a allelic form or fragment of the DNA sequences specified in (a) , (b) , (c) , (d) , or (e) . In a second aspect the invention relates to an isolated enzyme exhibiting beta-glucanase activity selected from the group consisting of:

(a) a polypeptide encoded by the beta-glucanase enzyme encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in Escherichia coli DSM 11342;

(b) a mature polypeptide having the mature part of an amino acid sequence as shown in SEQ ID NO 2;

(c) an analogue of the polypeptide defined in (a) or (b) which is at least 70 % homologous with said polypeptide; and

(d) an allelic form or fragment of (a) , (b) or (c) .

In a still further aspect the invention provides a recombinant expression vector, which enables heterologous recombinant production of an enzyme of the invention. Thereby it is possible to make a highly purified beta-glucanase composition, characterized in being free from homologous impurities. This is highly advantageous for a number of industrial applications. Finally the invention relates to an isolated substantially pure biological culture of the Escherichia coli strain DSM No. 11342 harbouring a beta-glucanase encoding DNA sequence (the beta-glucanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in Escherichia coli DSM 11342) obtained from a strain of the filamentous fungus Phaffia rhodozyma , or any mutant of said E . coli strain having retained the beta-glucanase encoding capability; and to an isolated substantially pure biological culture of the filamentous fungus Phaffia rhodozyma CBS No. 6938, from which the DNA sequence presented as SEQ ID No. 1 has been derived.

DEFINITIONS

Prior to discussing this invention in further detail, the following terms will first be defined. "A cloned DNA sequence" : The term "A cloned DNA sequence", refers to a DNA sequence cloned in accordance with standard cloning procedures used in genetic engineering to relocate a segment of DNA from its natural location to a different site where it will be reproduced. The cloning process involves excision and isolation of the desired DNA segment, insertion of the piece of DNA into the vector molecule and incorporation of the recombinant vector into a cell where multiple copies or clones of the DNA segment will be replicated. 5 The "cloned DNA sequence" of the invention may alternatively be termed "DNA construct" or "isolated DNA sequence" .

"Obtained from" : For the purpose of the present invention the term "obtained from" as used herein in connection with a lθ specific microbial source, means that the enzyme is produced by the specific source, or by a cell in which a gene from the source have been inserted.

"An isolated polypeptide" : As defined herein the term, "an isolated polypeptide" or "isolated beta-glucanase", as used

15 about the beta-glucanase of the invention, is a beta-glucanase or beta-glucanase part which is essentially free of other non- beta-glucanase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about

20 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.

The term "isolated polypeptide" may alternatively be termed "purified polypeptide" .

"Homologous impurities": As used herein the term "homologous

25 impurities" means any impurity (e.g. another polypeptide than the enzyme of the invention) which originate from the homologous cell where the enzyme of the invention is originally obtained from. In the present invention the homologous cell may e . g . be a strain of Phaffia rhodozyma .

30 "beta-glucanase encoding part": As used herein the term

"beta-glucanase encoding part" used in connection with a DNA sequence means the region of the DNA sequence which corresponds to the region which is translated into a polypeptide sequence.

In the DNA sequence shown in SEQ ID NO 1 it is the region

35 between the first "ATG" start codon ("AUG" codon in mRNA) and the following stop codon ("TAA", "TAG" or "TGA").

The translated polypeptide may further, in addition to the mature sequence exhibiting beta-glucanase activity, comprise an N-terminal signal sequence. The signal sequence generally guides the secretion of the polypeptide. For further information see Stryer, L. , "Biochemistry" W.H., Freeman and Company/New York, ISBN 0-7167-1920-7.

"beta-glucanase" is defined according to standard enzyme EC-classification as EC 3.2.1.6.

Official Name: ENDO-1, 3 (4) -BETA-GLUCANASE.

Reaction catalysed: ENDOHYDROLYSIS OF 1,3- OR 1,4-LINKAGES IN BETA-D-GLUCANS WHEN THE GLUCOSE RESIDUE WHOSE REDUCING GROUP IS INVOLVED IN THE LINKAGE TO BE HYDROLYSED IS ITSELF SUBSTITUTED AT C-3.

Cloned DNA sequence

In its first aspect the invention relates to a cloned DNA sequence encoding an enzyme exhibiting beta-glucanase activity, which DNA sequence comprises:

(a) the beta-glucanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in Escherichia coli DSM 11342;

(b) the DNA sequence shown in positions 1-1275 in SEQ ID NO 1 or its complementary strand;

(d) a DNA sequence which hybridizes with the DNA sequence shown in positions 1-1275 in SEQ ID NO 1 at low stringency; (e) a DNA sequence which, because of the degeneracy of the genetic code, does not hybridize with the sequences of (b) or (d) , but which codes for a polypeptide having exactly the same a ino acid sequences as the polypeptide encoded by these DNA sequences ; and (f) a DNA sequence which is a allelic form or fragment of the DNA sequences specified in (a) , (b) , (c) , (d) , or (e) .

In this specification, whenever reference is made to the beta-glucanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in DSM 11342 such reference is also intended to include the beta-glucanase encoding part of the DNA sequence presented in SEQ ID NO 1.

Accordingly, the terms "the beta-glucanase encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in DSM 11342" and "the beta-glucanase encoding part of the DNA sequence presented in SEQ ID NO 1" may be used interchangeably. The DNA sequence may be of genomic, cDNA, or synthetic origin or any combination thereof. The present invention also encompasses a cloned DNA sequence which encodes an enzyme exhibiting beta-glucanase activity having the amino acid sequence set forth as the mature part of SEQ ID NO 2 , which differ from SEQ ID NO 1 by virtue of the degeneracy of the genetic code. The DNA sequence shown in SEQ ID NO 1 and/or an analogue DNA sequence of the invention may be cloned from a strain of the filamentous fungus Phaffia rhodozyma producing the enzyme with beta-glucanase activity, or another or related organism as further described below (See section "Microbial sources"). Alternatively, the analogous sequence may be constructed on the basis of the DNA sequence presented as the beta- glucanase encoding part of SEQ ID No. 1, e . g . be a sub-sequence thereof, and/or be constructed by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the beta-glucanase 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 (i.e. a variant of the beta-glucanase of the invention) .

When carrying out nucleotide substitutions, amino acid changes are preferably of a minor nature, i.e. 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 extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification, such as a poly-histidine tract; an antigenic epitope or a binding domain. 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. For a general description of nucleotide substitution, see e . g . Ford et al., (1991), Protein Expression and Purification 2, 95-107.

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 polypeptide. Amino acids essential to the activity of the poly- peptide encoded by the cloned DNA sequence 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 (cf. e . g . 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. beta-glucanase) 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 analysis, crystallography or photo affinity labelling (cf. e.g. 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) .

Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include, ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter (s) and terminator.

The DNA sequence of the invention can be cloned from the strain Escherichia coli DSM No. 11342 using standard cloning techniques e . g . as described by Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab.; Cold Spring Harbor, NY.

The DNA sequence of the invention may also be cloned from an organism producing said enzyme, e.g. by purifying the enzyme, amino acid sequencing, and preparing a suitable probe or PCR primer based on this amino acid sequence.

The DNA sequence of the invention can also be cloned by any general method involving

• cloning, in suitable vectors, a cDNA library from any organism expected to produce the beta-glucanase of interest,

• transforming suitable yeast host cells with said vectors,

• culturing the host cells under suitable conditions to express any enzyme of interest encoded by a clone in the cDNA library,

• screening for positive clones by determining any beta- glucanase activity of the enzyme produced by such clones, and

• isolating the enzyme encoding DNA from such clones.

A general isolation method has been disclosed in WO 93/11249 and WO 94/14953, the contents of which are hereby incorporated by reference. A more detailed description of the screening method is given in a working example herein (vide infra) .

Alternatively, the DNA encoding a beta-glucanase of the invention may, in accordance .with well-known procedures, conveniently be cloned from a suitable source, such as any of organisms mentioned in the section "Microbial Sources", 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 beta- glucanase encoding part of the nucleotide sequences presented as SEQ ID No. 1 or any suitable subsequence thereof, or the basis of the amino acid sequence SEQ ID NO 2.

Homology of DNA sequences

The DNA sequence homology referred to above is determined as the degree of identity between 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 (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 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 preferably of at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97% with the beta-glucanase encoding part of the DNA sequence shown in SEQ ID No. 1.

Hybridization:

The hybridization referred to above is intended to comprise an analogous DNA sequence which hybridizes to a double-stranded nucleotide probe corresponding to the beta-glucanase encoding part of the DNA sequence shown in SEQ ID NO 1, i.e. nucleotides 1-1275, under at least low stringency conditions as described in detail below.

Suitable experimental conditions for determining hybridization at low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt ' s solution (Sambrook et al. 1989), 0.5 % SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a concentration of lOng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem . 132:6-13), ³²P-dCTP-labeled (specific activity > 1 x 10⁹ cpm/μg ) probe for 12 hours at ca . 45°C. The filter is then washed twice for 30 minutes in 2 x SSC, 0.5 % SDS at least 55 °C (low stringency) , more preferably at least 60°C (medium stringency) , still more preferably at least 65 °C (medium/high stringency) , even more preferably at least 70 °C (high stringency) , even more preferably at least 75 °C (very high stringency) .

Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using a x-ray film.

Homology to amino acid sequences:

The polypeptide homology referred to above is determined as the degree of identity between 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 (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453. Using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1, the mature part of a polypeptide encoded by an analogous DNA sequence exhibits a degree of identity preferably of at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, especially at least 97% with the mature part of the amino acid sequence shown in SEQ ID NO 2. The present invention is also directed to Beta- glucanase variants which have an amino acid sequence which differs by no more than three amino acids, preferably by no more than two amino acids, and more preferably by no more than one amino acid from the mature part of the amino acid sequence set forth in SEQ ID NO 2.

Immunological cross-reactivity

Antibodies to be used in determining immunological cross- reactivity may be prepared by using a purified beta-glucanase. More specifically, antiserum against the beta-glucanase 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, I munochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) . Purified immunoglobulins may be obtained from the antiserum obtained, for example by salt precipitation ((NH₄) S0 ) , followed by dialysis and ion exchange chromatography, e . g . on DEAE-Sephadex. Immunochemical characterization of proteins may be performed either by Outcherlony double-diffusion analysis

(O. Ouchterlony in: Handbook of Experimental Immunology (D.M.

Weir, Ed.), Blackwell Scientific Publications, 1967, pp. 655-

706), by crossed immunoelectrophoresis (N. Axelsen et al., supra. Chapters 3 and 4) , or by rocket immunoelectrophoresis

(N. Axelsen et al., Chapter 2).

Microbial Sources

At the priority date of the present invention, the taxonomy applied below are in accordance with the World Wide web (WWW)

NCBI taxonomy browser.

The beta-glucanase of and the corresponding cloned DNA sequence of the invention may be obtained from any fungal strain. A preferred genus is Phaffia , wherein a preferred strain is Phaffia rhodozyma .

An isolate of a strain of Phaffia rhodozyma from which an beta-glucanase of the invention can be obtained is Phaffia rhodozyma CBS No. 6938, which has been deposited according to the Budapest Treaty on the International Recognition of the

Deposit of Microorganisms for the Purposes of Patent Procedure at the Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740

AG Baarn, The Netherlands, (CBS) .

The expression plasmid pYES 2.0 comprising the full length cDNA sequence encoding the beta-glucanase of the invention has been transformed into a strain of the Escherichia coli which was deposited according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH. , Masheroder Weg lb, D- 38124 Braunschweig, Federal Republic of Germany, (DSM) . Deposit date : 18 of December 96

Depositor's ref. : NN049285

DSM No. : Escherichia coli DSM No. 11342

Expression vectors

The expression vector of the invention may be any expression vector that is conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which the vector it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent 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 expression vector, the DNA sequence encoding the beta-glucanase 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 which are either homologous or heterologous to the host cell. The procedures used to ligate the DNA sequences coding for the beta-glucanase, the promoter and the terminator and to insert them into suitable vectors are well known to persons skilled in the art (cf. e . g . Sambrook et al., (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY) .

Examples of suitable promoters for use in filamentous fungus host cells are, e.g. the ADH3 promoter (McKnight et al . , The EMBO J . (1985) , 2093 - 2099) or the tpiA promoter. Examples of other useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a- amylase, Aspergillus niger acid stable a-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (gluA) , Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase or Aspergillus nidulans acetamidase. Host Cells

The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. The term "host cell" encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur during replication.

The cell is preferably transformed with a vector comprising a nucleic acid sequence of the invention followed by integration of the vector into the host chromosome. "Transformation" means introducing a vector comprising a nucleic acid sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non-homologous recombination as described above. The choice of host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g. a prokaryote, or a non- unicellular microorganism, e.g. a eukaryote.

In a preferred embodiment, the host cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota,

Basidiomycota, Chytridiomycota , and Zygo ycota (as defined by Hawksworth et al . , In , Ainsworth and Bisby' s Dictionary of The Fungi , 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al . , 1995, supra , page 171) and all mitosporic fungi (Hawksworth et al . , 1995, supra) . Representative groups of Ascomycota include, e.g., Neurospora , Eupenicillium (=Penicillium) , Emericella (= Aspergillus) , Eurotium (=Aspergillus) , and the true yeasts listed above. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, e.g., Allomyces , Blastocladiella , Coelomomyces , and aquatic fungi. Representative groups of Oomycota include, e . g . , Saproleg- niomycetous aquatic fungi (water molds) such as Achlya . Examples of mitosporic fungi include Aspergillus , Penicillium , Candida , and Alternaria . Representative groups of Zygomycota include, e . g. , Rhizopus and Mucor.

In a preferred embodiment, the fungal host cell is a filamentous fungal cell. "Filamentous fungi" include all fila- 5 mentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al . , 1995, supra) . In a more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium , Aspergillus , Fusarium, Humicola , Mucor, Myceliophthora , Neurospora ,

10 Penicillium , Thielavia , Tolypocladium , and Trichoderma or a teleomorph or synonym thereof.

Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se . Suitable

15 procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al . , 1984, Proceedings of the National Academy of Sciences USA 81:1470-1474. A suitable method of transforming Fusarium species is described by Malardier et al . , 1989, Gene 78:147-156 or in copending US Serial No.

20 08/269,449. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al . , 1983, Journal of Bacteriology

25 153:163; and Hinnen et al . , 1978, Proceedings of the National Academy of Sciences USA 75:1920. Mammalian cells may be transformed by direct uptake using the calcium phosphate precipitation method of Graham and Van der Eb (1978, Virology 52:546) .

30

Method of producing beta-glucanase

The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence 35 encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture. When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme of the invention. Thereby it is possible to make a highly purified beta- glucanase composition, characterized in being free from homologous impurities .

In the present invention the homologous host cell may be a strain of Phaffia rhodozyma . The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed beta-glucanase may conveniently be secreted into the culture medium and may be recovered therefrom 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.

Activity of beta-glucanase of the invention:

The beta-glucanase of the invention has ENDO-1, 3 (4) -BETA- GLUCANASE (EC 3.2.1.6) enzyme activity.

This may be tested, as described in further details in example 2 herein (vide infra) , as a positive activity towards the substrates AZCL-β-Glucan or AZCL-curdlan, and a substantial negative activity towards AZCL-He-Cellulose.

Industrial applications of a beta-glucanase of the invention: It is presently believed that a beta-glucanase of the invention may advantageously be used in any of the general known applications for a beta-glucanase.

Examples are given below of preferred uses of the enzyme preparation of the invention. The dosage of the enzyme, either used as a single enzyme or in an composition, said composition comprising either other enzyme activities, or other ingredients according to the art, of the invention and other conditions under which the enzyme or composition is used may be determined on the basis of methods known in the art. Examples are use of the beta-glucanase of the invention in the brewing industry, as the enzymes degrades the barley b- glucan and thereby reduces the viscosity and improves the filterability of the wort. In brewing the high specificity for β- glucans is an advantage as compared to other endoglucanases as the viscosity caused by β-glucan can be reduced without simultaneous degradation of the cellulose structures which are essential for the filterability of the wrote where brewers spent grains act as filter-aid. Further the beta-glucanse according to the invention is preferably used as an agent for degradation or modification of b- glucan containing material such as microbial cell walls. In particular, the enzyme preparation of the invention may be used for rupturing or lysing cell walls of microorganisms thereby enabling recovery of desirable products produced by the microorganism.

It will be understood that the specific composition of the enzyme preparation to be used should be adapted to the composition of the cell wall to be ruptured or lysed. For instance, yeast cell walls have been found to comprise two main layers, an outer layer of protein-mannan complex and an inner glucan layer. In order to efficiently rupturing the cell wall of yeast it is desirable that the enzyme preparation comprises at least protease, mannanase and b-glucanase activity. The extract recovered after rupture of the microbial cell walls normally comprises a number of different components, such as pigments, vitamins, colorants and flavourants. Extracts obtained from rupture of yeast, i.e. yeast extracts, are used as such, e.g. for food or feed applications - or components thereof may be recovered and optionally further processed.

Examples of such products include vitamins, colorants (e.g. carotenoids, Q-10 and astaxanthin) , enzymes, proteins and flavour components or flavour enhancers (e.g. MSG, 5 ' -GMP and 5' -IMP). The products to be recovered may be inherent products of the microorganism in question, or may be products which the microorganism has been constructed to produce, e.g. recombinant products .

In addition the enzyme preparation of the invention may be used in the production of protoplasts from yeasts (e.g. of Saccharomyces sp. or Schizosaccharomyces sp.) or from fungi (e.g. Aspergillus sp. or Penicillium sp.). Preparation and regeneration of protoplast from such organisms are important in fusion, transformation and cloning studies. The production of protoplasts may be performed in accordance with methods known in the art.

The invention may also be used for improving fungal transformation.

Further, the enzyme or enzyme preparation according to the invention may be used in the preparation of pharmaceuticals, especially products entrapped inside the cells in the cytoplasmic membrane, the periplasmic space and/or the cell wall.

In addition, the enzyme preparation of the invention may be used in the modification of b-glucans such as curdlan and laminarin. Further an preferred use of an beta-glucanase of the invention is for use in the process of wine-making, in particular for removing of beta-glucans before filtration of the wine.

Such a use is described in further details in ("Industrial enzymes in the biotechnology of wine" International Food Technology No. 4/1990, Deutscher Fachverlag GmbH, Frankfurt, Germany; See especially p . 4 section "Beta-glucanases" and p . 5 section "conclusion" ; and "Wine microbiology and Biotechnology" (1993) , edited by Graham H. Fleet, see chapter 17 "Enzymes in winemaking" ) . Even further an preferred use of an beta-glucanase of the invention is for use in the process of juice-making, in particular for clarification of the juice.

Such a use is described in further details in ("Industrial enzymes in the biotechnology of wine" International Food Technology No. 4/1990, Deutscher Fachverlag GmbH, Frankfurt, Germany; See especially p .3 section "Clarification of the juice . . " , p . 4 section "Beta-glucanases" and p . 5 section "conclusion" ) .

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:

Phaffia rhodozyma CBS No. 6938 comprises the beta- glucanase encoding DNA sequence of the invention. Escherichia coli DSM 11342 containing the plasmid comprising the full length cDNA sequence, coding for the beta- glucanase of the invention, in the shuttle vector pYES 2.0.

Other strains: Yeast strain: The Saccharomyces cerevisiae strain used was W3124 (MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137; prcl::HIS3; prbl:: LEU2 ; cir+) .

E. coli strain: DH10B (Life Technologies)

Plasmids:

The Aspergillus expression vector pHD414 is a derivative of the plasmid p775 (described in EP 238 023) . The construction of pHD414 is further described in WO 93/11249.

pYES 2.0 (Invitrogen)

pA2BG171 (See example 1)

General molecular biology methods:

Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C R. , and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990).

Enzymes for DNA manipulations were used according to the specifications of the suppliers.

Enzymes for DNA manipulations

Unless otherwise mentioned all enzymes for DNA manipulations, such as e.g. restiction endonucleases, ligases etc., are obtained from New England Biolabs, Inc.

Expression cloning in yeast

Expression cloning in yeast was done as comprehensively described by H. Dalboege et al. (H. Dalboege et al Mol. Gen. Genet (1994) 243:253-260.; WO 93/11249; WO 94/14953), which are hereby incorporated as reference.

All individual steps of Extraction of total RNA, cDNA synthesis, Mung bean nuclease treatment, Blunt-ending with T4 DNA polymerase, and Construction of libraries was done according to the references mentioned above.

Fermentation procedure of Phaffia rhodozyma CBS No. 6938 for mRNA isolation: Fermentation of Phaffia rhodozyma CBS No. 6938 was performed in shake-flasks with BPX media (described below) at 20 °C for 2-3 days.

The resulting culture broth was filtered through miracloth and the mycelium was frozen down in liquid nitrogen. mRNA was isolated from mycelium from this culture as described in (H. Dalboege et al Mol. Gen. Genet (1994) 243:253-

260.; WO 93/11249; WO 94/14953).

Identification of positive yeast clones: Identification of positive yeast clones (i.e. clones which comprise a gene encoding for beta-glucanase activity) was done as described below.

The yeast transformants is plated on SC agar containing 0.1% AZCL beta-glucan (Megazyme, Australia) and 2% galactose and incubated for 3-5 days at 30°C

Beta-glucanase positive colonies is identified as colonies surrounded by a blue halo.

Isolation of a cDNA gene for expression in Aspergillus : A beta-glucanase-producing yeast colony is inoculated into 20 ml YPD broth in a 50 ml glass test tube. The tube is shaken for 2 days at 30°C The cells are harvested by centrifugation for 10 min. at 3000 rpm. DNA is isolated according to WO 94/14953 and dissolved in 50 ml water. The DNA is transformed into E . coli by standard procedures. Plasmid DNA is isolated from E . coli using standard procedures, and analyzed by restriction enzyme analysis. The cDNA insert is excised using appropriate restriction enzymes and ligated into an Aspergillus expression vector.

Transformation of Aspergillus oryzae or Aspergillus niger Protoplasts may be prepared as described in WO 95/02043, p. 16, line 21 - page 17, line 12, which is hereby incorporated by reference.

100 μl of protoplast suspension is mixed with 5-25 μg of the appropriate DNA in 10 μl of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH = 7.5, 10 mM CaCl ) . Protoplasts are mixed with pA2BG171 (See example 1) . The mixture is left at room temperature for 25 minutes. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl and 10 mM Tris-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85 ml of the same solution is added and carefully mixed. The mixture is left at room temperature for 25 minutes, spun at 2500 g for 15 minutes and the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one more sedimentation the protoplasts are spread on minimal plates (Cove, Biochem. Biophys. Acta 113 (1966) 51-56) containing 1.0 M sucrose, pH 7.0 , 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit background growth. After incubation for 4-7 days at 37 °C spores are picked and spread for single colonies. This procedure is repeated and spores of a single colony after the second reisolation is stored as a defined transformant .

Test of A . oryzae transformants

Each of the A . oryzae transformants are inoculated in 10 ml of YPM (cf. below) and propagated. After 2-5 days of incubation at 30°C, the supernatant is removed. The beta-glucanase activity is identified by applying 20 μl supernatant to 4 mm diameter holes punched out in agar plates containing 0.2% AZCL beta- glucan (Megazyme, Australia) . Beta-glucanase activity is then identified as a blue halo. Fed batch fermentation:

Fed batch fermentation was performed in a medium comprising maltodextrin as a carbon source, urea as a nitrogen source and yeast extract. The fed batch fermentation was performed by inoculating a shake flask culture of A. oryzae host cells in question into a medium comprising 3.5% of the carbon source and 0.5% of the nitrogen source. After 24 hours of cultivation at pH 7.0 and 34 °C the continuous supply of additional carbon and nitrogen sources were initiated. The carbon source was kept as the limiting factor and it was secured that oxygen was present in excess. The fed batch cultivation was continued for 4 days.

Shake-flask fermentation of A . oryzae :

Fermentation of A . oryzae was performed in shake-flasks with DAP-2C-1 media (described below) . The inoculation was performed by cultivation of A . oryzae in cove tubes containing 1,2 M sorbitol, 1 % glucose and 0,01 M urea at 37 °C for 4-5 days. The spores from cove tubes was dissolved in sterile water added 0,1 % tween and used to inoculate the shake-flasks. The fermentation was continued at 30 °C for 3 days.

Isolation of the DNA sequence shown in SEQ ID No. 1:

The beta-glucanase encoding part of the DNA sequence shown in SEQ ID No. 1 coding for the beta-glucanase of the invention can be obtained from the deposited organism Escherichia coli DSM 11342 by extraction of plasmid DNA by methods known in the art (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY).

Media

YPD: 10 g yeast extract, 20 g peptone, H 0 to 900 ml. Autoclaved, 100 ml 20% glucose (sterile filtered) added.

YPM: 10 g yeast extract, 20 g peptone, H₂0 to 900 ml. Autoclaved, 100 ml 20% maltodextrin (sterile filtered) added.

10 x Basal salt: 75 g yeast nitrogen base, 113 g succinic acid, 68 g NaOH, H₂0 ad 1000 ml, sterile filtered. SC-URA: 100 ml 10 x Basal salt, 28 ml 20% casamino acids without vitamins, 10 ml 1% tryptophan, H 0 ad 900 ml, autoclaved, 3.6 ml 5% threonine and 100 ml 20% glucose or 20% galactose added.

SC-agar: SC-URA, 20 g/1 agar added.

SC-variant agar: 20 g agar, 20 ml 10 x Basal salt, H₂0 ad 900 ml, autoclaved

AZCL beta-glucan (Megazyme, Australia)

PEG 4000 (polyethylene glycol, molecular weight = 4,000) (BDH, England)

BPX media: 50 g potato flour, 25 g barley flour, 0,05 g BAN 800 MG, H₂0 ad 800 mL. Inkubated at 60-85 °C for 30 min. 5 g Na- Caseinate at 60 °C added and dissolve. Thereafter 10 g soy grit, 4,5 g Na₂HP0₄12H₂0, 0,1 L Pluronic added, H₂0 ad 1000 mL. pH adjusted to 7,4-7,5 with 4 N NaOH. Autoclaved.

DAP-2C-1 media: 11 g MgSO₄7H₂0, 1 g KH₂P0₄, 2 g citric acid, 30 g maltodextrin, 6 g K₃PO 3H₂0, 0,5 g yeast extract (Difco) , 1 mL pluronic, 0,5 mL KU-6 (see below) , H₂0 ad 1000 mL. 1 CaC0₃ tablet per 250 mg added. Autoclaved, 3,5 mL 50 % (NH₄) HPO ₄ and 5 mL 20 % lactic acid (sterile filtered) added. pH is 5,2. KU-6: 6,8 g ZnCl₂, 2,5 g CuS0₄5H₂0, 0,24 g NiCl₂6H₂0, 13,9 g FeS07H₂0, 8,45 g MnS0₄H₂0, 3 g citric acid, H₂0 ad 1000 mL. Autoclaved.

EXAMPLES

EXAMPLE 1

Cloning and expression of a beta-glucanase from Phaffia rhodozyma CBS No. 6938 Cloning and expression was done by using the Expression cloning in yeast technique as described above. mRNA was isolated from Phaffia rhodozyma , CBS No. 6938, grown as described above with agitation to ensure sufficient aeration. Mycelia were harvested after 2-3 days' growth, immediately frozen in liquid nitrogen and stored at -80°C A library from Phaffia rhodozyma , CBS No. 6938, consisting of approx. 4xl0⁵ individual clones was constructed in E . coli as described with a vector background of 1%. Plasmid DNA from some of the pools was transformed into yeast, and 50-100 plates containing 250-400 yeast colonies were obtained from each pool.

Beta-glucanase-positive colonies were identified and isolated on SC-agar plates as described above (vide supra) . cDNA inserts were amplified directly from the yeast colonies and characterized as described in the Materials and Methods section above. The DNA sequence of the cDNA encoding the beta- glucanase is shown in SEQ ID No. 1 and the corresponding amino acid sequence is shown in SEQ ID No. 2. In SEQ ID No. 1 DNA nucleotides from No 1 to No. 1275 define the beta-glucanase encoding region.

The cDNA is obtainable from the plasmid in DSM 11342. Total DNA was isolated from a yeast colony and plasmid DNA was rescued by transformation of E . coli as described above. In order to express the beta-glucanase in Aspergillus , the DNA was digested with appropriate restriction enzymes, size fractionated on gel, and a fragment corresponding to the beta- glucanase gene was purified. The gene was subsequently ligated to pHD414, digested with appropriate restriction enzymes, resulting in the plasmid pA2BG171. After amplification of the DNA in E . coli the plasmid was transformed into Aspergillus oryzae as described above.

Test of A . oryzae transformants

Each of the transformants were tested for enzyme activity as described above. Some of the transformants had beta-glucanase activity which was significantly larger than the Aspergillus oryzae background. This demonstrates efficient expression of the beta-glucanase in Aspergillus oryzae . EXAMPLE 2 :

Assay to test for ENDO-1,3 (4) -BETA-GLUCANASE (EC 3.2.1.6) enzyme activity.

Substrates: 0.1 w/v% AZCL-β-Glucan; 0.1 w/v% AZCL-curdlan; 0.1 w/v% AZCL-He-Cellulose. (all from MegaZyme, Australia) .

General assay conditions: Shaking waterbath, 37 °C for 1 hour.

Incubation mixture: 0.5 ml supernatant + 0.5 ml 0.1 w/v% subtrate

+ 0.5 ml acetate-buffer (pH 4.0). Substate was also dissolved in the acetatebuffer. The reaction was stopped with 3 ml of stop reagent. The insoluble curdlan was spun down by centrifugation and the dissolved blue coloured part determined on a Hitachi

Spectrophotometer at 590nm, 1 cm.

Stop reagent:

Sodiumacetate 40 gram

Zinkacetate 4 gram Deionized water to 200 ml

The pH was adjusted to 5.0 before mixing with 800 ml of 2- methoxyethanol .

0.5 ml supernatant from a fed batch fermentation of an A. Orytzae strain transformed with pA2BG171 plasmid (see Example 1) was tested as described above using the substrates AZCL-β-Glucan; AZCL-curdlan; and AZCL-He-Cellulose.

Positive results were obtained for the substrates AZCL-β-Glucan and AZCL-curdlan, and no significant activity was detected for the substrate AZCL-He-Cellulose.

The substrate AZCL-He-Cellulose comprises substantially only 1,4-bindings in β-D-glycanes, where the other two comprise 1,3- bindings in β-D-glycanes.

This demonstrates that an β-glucanase of the invention has ENDO-1, 3 (4) -BETA-GLUCANASE (EC 3.2.1.6) activity. SEQUENCE LISTING

SEQ ID No. 1 shows a cloned DNA sequence of the invention, comprising a DNA sequence encoding an enzyme exhibiting beta-glucanase activity.

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1275 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: Phaffia rhodozyma

(B) STRAIN: CBS No. 6938 (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1275

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATG CAT CTT GCC AAC GTC CTT CTT ACC CTT CTT CCC GTG TCA TTA TTG 48 Met His Leu Ala Asn Val Leu Leu Thr Leu Leu Pro Val Ser Leu Leu 1 5 10 15

GCT ACA GAA TCC TTG GCT GGT TCT AGT TCC CAC TCG GCA CAC GCG CTT 96 Ala Thr Glu Ser Leu Ala Gly Ser Ser Ser His Ser Ala His Ala Leu 20 25 30

CCC GCC AGA CGT CGT CAC AAC AAA GGC AGA GCG CTT TCC CCC ATC AAG 144 Pro Ala Arg Arg Arg His Asn Lys Gly Arg Ala Leu Ser Pro lie Lys 35 40 45

GCT TCA AAT TCT TCC TCC GAG CAC GAG ACG AAC CGA ATC GCT AGC GCT 192 Ala Ser Asn Ser Ser Ser Glu His Glu Thr Asn Arg lie Ala Ser Ala 50 55 60

GGC GCT AGT GCA GAT GAC TTT TCA CCA GTC ACC GGT AGA CGG GTC AGT 240 Gly Ala Ser Ala Asp Asp Phe Ser Pro Val Thr Gly Arg Arg Val Ser 65 70 75 80

AAA CGG GCT CAG TGC GGA GTC TCT TCA CCG GCG ACA TCA TCC AAA ACA 288 Lys Arg Ala Gin Cys Gly Val Ser Ser Pro Ala Thr Ser Ser Lys Thr 85 90 95 TCA TCT ACC ATC ACT GTC GGA GCA GCC GTT GTC CCA ACT GCA GCC GCG 336 Ser Ser Thr lie Thr Val Gly Ala Ala Val Val Pro Thr Ala Ala Ala 100 105 110

ACC TCC AGC TCG AAA TGG AAG CTT GAT CTC GAA GCA AAA GGA AAT TCC 384 Thr Ser Ser Ser Lys Trp Lys Leu Asp Leu Glu Ala Lys Gly Asn Ser 115 120 125

TTC TTC GAC ACC TTC AAC TTC TGG GCC TAT GAC GAC CCC ACA CAT GGG 432 Phe Phe Asp Thr Phe Asn Phe Trp Ala Tyr Asp Asp Pro Thr His Gly 130 135 140

ACC GTC ACT TAT GTC AGT CAA GAT GAG GCT ACA AAG TCC AAC CTA GCC 480 Thr Val Thr Tyr Val Ser Gin Asp Glu Ala Thr Lys Ser Asn Leu Ala 145 150 155 160

ACG GTC AAC GGA AAA GGA AAT GCT GTC TTG GCG GTC GAC ACC ACT CAG 528 Thr Val Asn Gly Lys Gly Asn Ala Val Leu Ala Val Asp Thr Thr Gin 165 170 175

AAT GTC CAG AAA GGC CGA AAG GCT GTT CGA CTT CAT TCG TCT TAC ATC 576 Asn Val Gin Lys Gly Arg Lys Ala Val Arg Leu His Ser Ser Tyr lie 180 185 190

TTC AAC GGA GGA CTC ATC CTT GCC GAT ATC GTT CAC ATG CCC ACC GGT 624 Phe Asn Gly Gly Leu lie Leu Ala Asp lie Val His Met Pro Thr Gly 195 200 205

TGT GGA ACA TGG CCT GCT TGG TGG TCC AAC GGA CCT GAC TGG CCC AAC 672 Cys Gly Thr Trp Pro Ala Trp Trp Ser Asn Gly Pro Asp Trp Pro Asn 210 215 220

AAA GGT GAG ATC GAC ATC CTC GAG GGT ACC CAT AGT TGG GAC AGA AAT 720 Lys Gly Glu lie Asp lie Leu Glu Gly Thr His Ser Trp Asp Arg Asn 225 230 235 240

CAG GTT TCT GTT CAC ACT AGT GAT GGA TGT ACG ATC CCT TCC AAC TAC 768 Gin Val Ser Val His Thr Ser Asp Gly Cys Thr lie Pro Ser Asn Tyr 245 250 255

GGA GCC TCG GCC GTG TTG ACG ACC GGA AGC TTT GTG AAC ACC AAT TGC 816 Gly Ala Ser Ala Val Leu Thr Thr Gly Ser Phe Val Asn Thr Asn Cys 260 265 270 GCC AGT TAC GCA ACG AGC AAC CAA GGG TGT GGC CAA CGA GAG TCA GCC 864 Ala Ser Tyr Ala Thr Ser Asn Gin Gly Cys Gly Gin Arg Glu Ser Ala 275 280 285

AGC CAC CAG GCT TAC GGC GAG CCG TTC AAT CAG AAC GGC GGC GGT GTG 912 Ser His Gin Ala Tyr Gly Glu Pro Phe Asn Gin Asn Gly Gly Gly Val 290 295 300

TAC GCC ATG AAG TGG GAT ACA TCC GGG ATC TCT GTC TAC TTC TTC CCT 960 Tyr Ala Met Lys Trp Asp Thr Ser Gly lie Ser Val Tyr Phe Phe Pro 305 310 315 320

AGA AAC GCC ATC CCG GCC GAT ATC ACC CAG GGT GTG CCC TTG CCG GAA 1008 Arg Asn Ala lie Pro Ala Asp lie Thr Gin Gly Val Pro Leu Pro Glu 325 330 335

ACT TGG GGA ACT CCT ATG GGT AAC TTC CCA TCG ACG TCT TGC GAA CCA 1056 Thr Trp Gly Thr Pro Met Gly Asn Phe Pro Ser Thr Ser Cys Glu Pro 340 345 350

TTC AAG TTC TTC AAA GAT CAC CAC ACC ATC ATC AAC ACC ACG TTC TGT 1104 Phe Lys Phe Phe Lys Asp His His Thr lie lie Asn Thr Thr Phe Cys 355 360 365

GGA GAC TGG GCC AAT TCA GAT TGG TGG ACG GCT GGG TCG GCC GGA AAC 1152 Gly Asp Trp Ala Asn Ser Asp Trp Trp Thr Ala Gly Ser Ala Gly Asn 370 375 380

GGT CAA TCT TGC GCC GCA AAG ACT GGA TAC AAC TCA TGC TCG GAT TAC 1200 Gly Gin Ser Cys Ala Ala Lys Thr Gly Tyr Asn Ser Cys Ser Asp Tyr 385 390 395 400

GTC CTC AAC AAT GGA GAC AAG TTT CAC GAA GCA TAT TGG GAG TTT GCG 1248 Val Leu Asn Asn Gly Asp Lys Phe His Glu Ala Tyr Trp Glu Phe Ala 405 410 415

TCT GTC AAG TAT TAT CAG CCG AAA TAG 1275

Ser Val Lys Tyr Tyr Gin Pro Lys * 420 425 SEQ ID No. 2 shows the amino acid sequence of a beta-glucanase of the invention.

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 424 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met His Leu Ala Asn Val Leu Leu Thr Leu Leu Pro Val Ser Leu Leu 1 5 10 15

Ala Thr Glu Ser Leu Ala Gly Ser Ser Ser His Ser Ala His Ala Leu 20 25 30

Pro Ala Arg Arg Arg His Asn Lys Gly Arg Ala Leu Ser Pro lie Lys 35 40 45

Ala Ser Asn Ser Ser Ser Glu His Glu Thr Asn Arg lie Ala Ser Ala 50 55 60

Gly Ala Ser Ala Asp Asp Phe Ser Pro Val Thr Gly Arg Arg Val Ser 65 70 75 80

Lys Arg Ala Gin Cys Gly Val Ser Ser Pro Ala Thr Ser Ser Lys Thr 85 90 95

Ser Ser Thr lie Thr Val Gly Ala Ala Val Val Pro Thr Ala Ala Ala 100 105 110

Thr Ser Ser Ser Lys Trp Lys Leu Asp Leu Glu Ala Lys Gly Asn Ser 115 120 125

Phe Phe Asp Thr Phe Asn Phe Trp Ala Tyr Asp Asp Pro Thr His Gly 130 135 140

Thr Val Thr Tyr Val Ser Gin Asp Glu Ala Thr Lys Ser Asn Leu Ala 145 150 155 160

Thr Val Asn Gly Lys Gly Asn Ala Val Leu Ala Val Asp Thr Thr Gin 165 170 175

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

Cys Gly Thr Trp Pro Ala Trp Trp Ser Asn Gly Pro Asp Trp Pro Asn 210 215 220

Lys Gly Glu lie Asp lie Leu Glu Gly Thr His Ser Trp Asp Arg Asn 225 230 235 240

Gin Val Ser Val His Thr Ser Asp Gly Cys Thr lie Pro Ser Asn Tyr 245 250 255

Gly Ala Ser Ala Val Leu Thr Thr Gly Ser Phe Val Asn Thr Asn Cys 260 265 270

Ala Ser Tyr Ala Thr Ser Asn Gin Gly Cys Gly Gin Arg Glu Ser Ala 275 280 285

Ser His Gin Ala Tyr Gly Glu Pro Phe Asn Gin Asn Gly Gly Gly Val 290 295 300

Tyr Ala Met Lys Trp Asp Thr Ser Gly lie Ser Val Tyr Phe Phe Pro 305 310 315 320

Arg Asn Ala lie Pro Ala Asp lie Thr Gin Gly Val Pro Leu Pro Glu 325 330 335

Thr Trp Gly Thr Pro Met Gly Asn Phe Pro Ser Thr Ser Cys Glu Pro 340 345 350

Phe Lys Phe Phe Lys Asp His His Thr lie lie Asn Thr Thr Phe Cys 355 360 365

Gly Asp Trp Ala Asn Ser Asp Trp Trp Thr Ala Gly Ser Ala Gly Asn 370 375 380

Gly Gin Ser Cys Ala Ala Lys Thr Gly Tyr Asn Ser Cys Ser Asp Tyr 385 390 395 400

Val Leu Asn Asn Gly Asp Lys Phe His Glu Ala Tyr Trp Glu Phe Ala 405 410 415

Ser Val Lys Tyr Tyr Gin Pro Lys * 420 425

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 136;_)

A. The indications made below relate to the microorganism referred to in the description

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet

Name of depositary institution

DEUTSCHE SAMMLUNG VON MKROORGA SMEN UND ZELLKULTUREN GmbH

Address of depositary institution (including postal code and country) Mascheroder Weg lb, D-38124 Braunschweig, GERMANY

Date of deposit Accession Number 18 December 199Λ DSM 11342

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information i q n_n_ i nued or) __Q a HH i +^• i nn a 1 chco r I — — — J

Until the publication of the mention of grant of a European patent or, where applicable, for twenty years from the date of filing if the application has been refused, withdrawn or deemed withdrawn, a sainple of the deposited microorganism is only to be provided to an mdependent expert nominated by the person requesting the sample (cf Rule 28(4) EPC) And as far as Australia is concerned, the expert option is likewise requested, reference bemg had to Regulation 3 25 of Australia Statutory Rules 1991 No 71 Also, for Canada we request that only an independent expert nominated by the Commissioner is authorized to have access to a sample of the microorganism deposited

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications e g , "Accession Number of Deposit")

Claims

1. A cloned DNA sequence encoding an enzyme exhibiting beta- glucanase activity, which DNA sequence is selected from the group comprising of:

(f) a DNA sequence which is a allelic form or fragment of the DNA sequences specified in (a) , (b) , (c) , (d) , or (e) .

2. An isolated enzyme exhibiting beta-glucanase activity selec- ted from the group comprising of: a) a polypeptide encoded by the beta-glucanase enzyme encoding part of the DNA sequence cloned into plasmid pYES 2.0 present in Escherichia coli DSM 11342;

an allelic form or fragment of (a) , (b) or (c) .

3. A recombinant expression vector comprising a cloned DNA sequence according to claim 1.

4. A host cell comprising a cloned DNA sequence according to any of claim 1 or a recombinant expression vector according to claim 3.

5. A method of producing an enzyme exhibiting beta-glucanase 5 activity, the method comprising culturing a cell according to 4 under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.

6. An isolated enzyme exhibiting beta-glucanase activity, 0 characterized in (i) being free from homologous impurities and

(ii) said enzyme is produced by the method according to claim 5.

7. A composition comprising the enzyme according to claim 2 or 5 6.

8. Use of an enzyme according to claim 2 or 6 or an enzyme composition according to claims 7 for the modification or degradation of a b-glucan containing material. 0

9. The use according to claim 8 , in which the enzyme or enzyme composition is used in the brewing industry.

10. The use according to claim 8, in which the enzyme or enzyme 5 composition is used in the preparation of protoplast.

11. The use of an enzyme according to claim 8, in which the enzyme or enzyme composition is used in the preparation of pigments, colorants, flavorants, yeast extracts.

30

12. The use of an enzyme according to claim 8, in which the enzyme or enzyme composition is used in the preparation of pharmaceuticals.

35 13. The use of an enzyme according to claim 8, in which the enzyme or enzyme composition is used in pharmaceuticals.

14. The use of an enzyme according to claim 8 , in which the enzyme or enzyme composition is used in a process of wine-making, in particular for removing of beta-glucans before filtration of the wine.

15. The use of an enzyme according to claim 8, in which the enzy- me or enzyme composition is used for the process of juice-making, in particular for clarification of the juice.

16. An isolated substantially pure biological culture of the deposited strain Escherichia coli DSM No. 11342.