A NOVEL ENZYME WITH beta -1,3-GLUCANASE ACTIVITY

Abstract: The present invention relates to a DNA construct exhibiting beta -1,3-glucanase activity, which DNA sequence: a) comprises the DNA sequence shown in SEQ ID no. 1, or b) comprises an analogue of the DNA sequence shown in SEQ ID no. 1, which (i) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1, and (ii) 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 (iii) encodes a polypeptide which is immunologically reactive with an antibody raised against a purified beta -1,3-glucanase encoded by the DNA sequence shown in SEQ ID No. 1 derived from Oerskovia Xanthineolytica LLG109. Further the invention relates to an expression vector comprising said DNA construct, a cell harbouring said DNA construct or expression vector, a method of producing the novel enzyme, a novel enzyme and preparation containing said novel enzyme, the use of the enzym for degrading or modification of beta -glucan containing material.

Description: Title: A novel enzyme with 0-1,3-glucanase activity FIELD OF THE INVENTION The present invention relates to a novel enzyme exhibiting β- 1,3-glucanase activity. More specifically a DNA construct encoding the novel enzyme, an expression vector comprising said DNA construct, a cell comprising said DNA construct or said recombinant expression vector, a method of producing said novel enzyme, an enzyme preparation comprising said novel enzyme, and use of the enzyme for degradation or modification of β-glucan containing material. BACKGROUND OF THE INVENTION A large diverse group of nonmotile, heterotrophic, eukaryotic organisms are collectively referred to as fungi. Most fungi are saprophytes, i.e. securing their food from dead organic material. Because of their heterotrophic properties, i.e. using organic material as carbon source, many fungi produce metabolites of industrial interest. Certain species are further useful as sources for food, whereas others are responsible for spoiling almost any organic material in which they come in contact. Fungal cells range in size from microscopic unicellular organ¬ isms to macroscopic as e.g. mushrooms. True fungi are in general terrestrial and includes Zygomycetes , such as Rhizopus , Basidiomycetes such as Puccinxa graminis , Ascomycetes , such as Neurospora and Saccharomyces and _->eι-te--O-r.ycetes, such as Penici Ilium and Aspergillus . Fungal cells Fungal microorganisms include multicellular as well as unicel¬ lular organisms and are in general considered to consist of yeasts and molds. Unicellular fungi are primarily yeasts, while the term mold is used for fungi that are predominantly mycelial. Fungal cells have a quite complex structure, constituting of a cytoplasm, comprising nucleus, mitochondria, microbodies etc. encapsulated by a cytoplasmic membrane. Chemically and structurally the cytoplasmic membrane consist of a bilayer of phospholipids with different proteins inserted into it. The cytoplasmic membrane is surrounded by the rigid cell wall. Structure of funσal cell walls The cell walls of most true fungal microorganisms contain a network of glucan, which gives the cell wall strength. Further major fungal cell walls constituents are mannoprotein and chitin. Glucan and chitin is far more resistant to microbial degrada¬ tion than cellulose, which is the major constituent of the cell wall of many fungi-like organisms, such as Oomycetes . Glucan is predominantly 0-1,3-linked with some branching via 1,6-linkage (Manners et al., Biotechnol. Bioeng, 38, p. 977, 1973) , and is known to be degradable by certain 3-1,3-glucanase systems. /8-1,3-glucanase includes the group of endo-jS-1,3-glucanases also called laminarinases (E.C. 3.2.1.39 and E.C. 3.2.1.6, Enzyme Nomenclature, Academic Press, Inc, 1992) . Pegg et al., Physiol. Plant Pathol., 21, p. 389-409, 1982, showed that a purified endo-j8-l,3-glucanase from tomato in combination with an exo-/3-l,3-glucanase of fungal origin were capable of hydrolysing isolated cell wall of the fungus Verticill ium alboatrum. Further, Keen et al., Plant Physiol., 71, p. 460-465 showed that a purified j8-l,3-glucanase from soy bean was capable of degrading isolated cell walls of fungi. Larσe scale degradation of the funσal cell wall The unit operation of cell disruption appears as an essential first step for intracellular products separation and downstream processing of valuable intracellular products. Large scale cell disruption is in general carried out by rather vigorous treatment involving the use of strong chemicals and/or mechanical means. This leaves the target protein with a very complex mixture of contaminants. In this context extensive industrial implementation of alterna¬ tive approaches to conventional microbial cell disruption techniques are becoming of increasing relevance (Asenjo et al., Bio/technol, 11, p. 214, 1993; De la Fuente et al., Appl. Microbiol. Biotechnol, 38, p. 763, 1993). Selective Cell Permeabilization (SCP) and Selective Protein Recovery (SPR) as a mean to increase bio-process productivity, economy and product quality by simplifying the downstream processing of intracellular products have proved to be very attractive in terms of their delicacy and specificity (Asenjo et al., 1993, supra ; Shen et al., Gene, 84, p. 1989). SCP and SPR involves the use of pure preparations of cell-wall- degrading 3-glucanases to increase fungal cell wall porosity (with very limited cell lysis) and facilitate the release of intracellular proteins. In this way, SCP gives primary separ¬ ation of the target product from some of its major contaminants. A major limitation to this approach is the relatively low level of expression of yeast lytic enzymes presently obtained in the bacteria used for the production of these enzymes (e.g. Oerskovia xanthineolytica, Andrews and Asenjo, Biotech. Bioeng, 30, p. 628, 1987). Today Oerskovia xanthineolytica are sometimes referred to as Cellulomonas cellulans . However, the name Oerskovia xanthineolytica will be used below. A number of commercial enzyme compositions useful in the enzymatic lysis of fungal cells are available. Such products normally comprise multiple enzymatic activities, e . g. including 0-1,3- and 0-1,6-glucanase, protease, chitinase, mannase and other enzymes capable of degrading cell wall components. The lvtic system of Oerskovia xanthineolytica LLG109 The lytic enzyme system of Oerskovia xanthineolytica LLG109 has partially been isolated and purified and some of the glucanase and protease components have been characterised (Ventom and Asenjo, Enzyme Microb. Technol., 13, p. 71, 1991). Although a single molecular species of lytic 0-1,3-glucanase has been characterized from O. xanthineolytica LLG109, most Oerskovia strains seem to have multiple 0-1,3-glucanase systems (Doi and Doi, J. Bacteriol., 168, p. 1272, 1986). While all observed molecular forms of these enzymes possess hydrolytic activity towards 0-1,3-glucans (0-1,3-glucanase activity) , only some are found capable of readily solubilizing yeast glucan and inducing lysis of viable yeast cells. In contrast, other types of endo-0-l,3-glucanases would solubilize yeast glucan only partially, causing only limited cell lysis (Doi and Doi, supra, 1986) . However, this multitude of enzyme species produced by Oerskovia may be partially due to proteolytic processing. The genetic relationship between these enzymes is still unclear, as the number of yeast lytic enzymes so far cloned is very limited. As a result, there is still limited knowledge about the gene structure and protein function relationship (Shen et al, J. Biol. Chem, 266, p. 1058, 1991; Shimol and Tademura, J. Bioche , 110, 608, 1991; Watanabe et al., J. Bacteriol, 174(1), p. 186, 1992; Yamamoto et al., Biosci. Biotechnol. Biochem. , 57, p. 1518-1525, 1993). Characterization of yeast lytic enzymes from O . xanthineolytica A purified lytic 0-1,3-glucanase showed a molecular mass of about 31 kDa, when estimated by SDS-PAGE and a pi of 5.0. The amino acid composition was also determined. The yield was optimized by the continuous culture process, but yields were still low. The specific activity of the enzyme was 11.1 U/mg. The K ^ for 0-1,3-glucanase activity on yeast glucan was 2.5 mg/ml for laminarin (a soluble 0-1,3-glucan) 0.95 mg/ml. The pH optimum for 0-1,3-glucanase was 8.0 on yeast glucan and 6.0 on laminarin substrate. The lytic 0-1,3-glucanase caused only limited lytic activity on viable yeast (Saccharomyces cerevisiae) cells (Ventom and Asenjo, supra , 1991) , but this was stimulated synergistically by the lytic protease component. In addition, there was detected another 0-1,3-glucanase component in clarified O . xanthineolytica continuous fermenta¬ tion broth, although it was not purified to homogeneity and subsequently characterized. Patent documents A number of 0-1,3-glucanase genes and uses thereof have been sought patented. An example is DD 226012 (Akad. Wissenshaft DDR) which concerns a method for production of a Bacillus 0-1,3-glucanase. Further, JP 61040792 A (DOI K) describes a cell wall-cytolase 0-1,3-glucanase recombinant plasmid for removing the cell walls of yeast. The gene is derived from Arthrobacter and is trans¬ formed into Escherichia group bacteria. EP 440.304 concerns plants provided with improved resistance against pathogenic fungi transformed with at least one gene encoding an intracellular chitinase, or an intra- or extracellular 0-1,3-glucanase. The matching recombinant polynucleotides is also disclosed. WO 87/01388 describes a method for preparing cell lytic enzymes, such as 0-1,3-glucanases, which can be produced by Oerskovia . WO 92/03557 discloses a recombinant DNA expression vector comprising a 2.7 kb DNA sequence, derived from Oerskovia xanthineolytica , encoding a 0-1,3-glucanase. Said glucanase, expressed in E. coli, exhibits glucanase activity and no protease activity. E. coli has a number of deficiencies in connection with large scale industrial enzyme production. First of all the glucanase is expressed intercellular. Consequently the cells need to be opened to get access to the enzyme. This makes recovery of the enzyme cumbersome and expensive. From WO 92/16632 a recombinant DNA sequence coding for a novel protein with 0-1,3-glucanase activity is known. The gene is derived from soy. Discussion of prior art Most presently available enzyme preparations for the use of lysing fungal cells contain protease activity, which leaves the lysed target protein with a very complex mixture of contaminants. It is therefore desirable to provide a 0-1,3-glucanase substantially free of protease activity which is capable of opening the cell walls in a gentle way. This will facilitate the recovery and purification of the target protein. Further, it would be advantageous to express the gene encoding the target protein in a heterologous host cell capable of increasing the production yield. SUMMARY OF THE INVENTION The present inventors have surprisingly succeeded in solving some of the above mentioned problems by providing a novel 0- 1,3-glucanase. First of all they have isolated and characterized a novel DNA sequence encoding a novel 26 kDa enzyme exhibiting 0-1,3- glucanase activity. According to the invention it is possible to prepare a novel single-component 0-1,3-glucanase. Accordingly, in a first aspect the invention relates to a DNA construct comprising a DNA sequence encoding a novel enzyme exhibiting 0-1,3-glucanase activity, which DNA sequence a) comprises the DNA sequence shown in SEQ ID No. l, or b) comprises an analogue of the DNA sequence shown in SEQ ID No. 1 , which i) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1, and ii) 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 iii) encodes a polypeptide which is immunologically reactive with an antibody raised against a purified 0-1,3-glucanase encoded by the DNA sequence shown in SEQ ID No. 1 derived from Oerskovia xanthineolytica LLG109. The Oerskovia xanthineolytica LLG109 strain has been deposited by the inventors according to the Budapest Treaty at the Deutshe Sammlung von Mikroorganis en und Zellkulturen GmbH., (DSM) . In the present context the expression "analogue" of the DNA sequence shown in SEQ ID No. 1 is intended to indicate any DNA sequence encoding an enzyme exhibiting 0-1,3-glucanase activity which has the properties i)-iii) above. Typically, the analog¬ ous DNA sequence - is isolated from another or related (e.g. the same) organism known or contemplated to produce the enzyme with 0-1,3- glucanase activity on the basis of any of the DNA sequences shown in SEQ ID No. 1, e .g. using the procedures described herein, or - is constructed on the basis of the DNA sequence shown in SEQ ID No. 1, e.g. by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the 0- 1,3-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 do give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to a 0-1,3-glucanase mutant with different properties than the native enzyme. Other examples of possible modifica¬ tions are insertion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence, or deletion of one or more nucleotides at either end or within the sequence. For instance, the analogous DNA sequence may be a subsequence of the DNA sequences shown in SEQ ID No. 1, for which the encoded protein exhibits 0-1,3- glucanase activity. The hybridization referred to in i) above is intended to indicate that the analogous DNA sequence hybridizes to the same probe as the DNA sequence encoding the novel 26 kDa 0-1,3- glucanase enzyme 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 60% homologous to the sequence shown above encoding a 0-1,3-glucanase of the inven¬ tion, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or even at least 95% homologous to the sequence shown in SEQ ID no. 1 below. The degree of homology referred to in ii) 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, 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 60%, such as at least 75% or 90% with the enzyme encoded by a DNA construct comprising the DNA sequence SEQ ID No. 1. The term "derived from" in connection with property iii) above is intended not only to indicate a 0-1,3-glucanase produced by Oerskovia xanthineolytica LLG109, but also a 0-1,3-glucanase encoded by a DNA sequence isolated from Oerskovia xanthineolytica LLG109 and produced in a host organism trans- formed with a vector comprising said DNA sequence. The immunological reactivity may be determined by the method described in the Materials and Methods section below. In a further aspect, the invention relates to the construction of an expression vector harbouring a DNA construct of the invention, a cell comprising the DNA construct or expression vector, and a method of producing a novel enzyme exhibiting 0- 1,3-glucanase activity, which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture. Still in a further aspect, the invention relates to a novel enzyme exhibiting 0-1,3-glucanase 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 0-1,3-glucanase encoded by the DNA sequence shown in SEQ ID No. 1 derived from Oerskovia xanthineolytica LLG109. The present invention also relates to an enzyme preparation useful for the degradation or modification of 0-glucan contain¬ ing materials, in particular icrobial cell wall material, said composition being enriched by an enzyme exhibiting 0-1,3,- glucanase activity, as described above. Finally contemplated according to the invention is the use of the novel enzyme or the enzyme preparation for preparation of fungal protoplasts or fungal extracts, especially yeast extracts. The final object of the invention is to provide a process for recovery of biological components from fungal cells, by subjecting the fungal cells in question to the novel 0-1,3- glucanase or an enzyme preparation thereof. BRIEF DESCRIPTION OF THE DRAWING Figure l shows the map of plasmid pPFFl. Figure 2 shows the map of plasmid pPF8A. Figure 3 shows the 1.5 kb BamHI-Kpnl fragment sequence accord¬ ing to the invention with compared with the predicted amino acid sequence and the partially determined amino acid sequence, Figure 4 shows the map of plasmid pPF15BK. Figure 5 shows the map of plasmid pPFUBgK. DETAILED DESCRIPTION OF THE INVENTION The present inventors have now surprisingly succeeded in providing a novel 0-1,3-glucanase substantially free of protease activity. By using the novel 0-1,3-glucanase to get access to the desired intercellular component facilitates the recovery process. The novel enzyme opens up the cells in a gently way, which involves making large and porous pores in the cell walls. This will further lead to a reduced amount of contaminants. Furthermore, the inventors have accomplished to express the gene encoding the novel 0-1,3-glucanase in a suitable heterologous host cell. Specifically in a strain of Bacillus subtil is , suitable for large scale industrial production. The yield of the novel 0-1,3-glucanase is increased in comparison to the parent cell from which the gene originates. The DNA sequence of the invention encoding an enzyme exhibiting 0-1,3-glucanase activity may be isolated by a general method involving - cloning, in suitable vectors, a DNA construct from Oerskovia Xanthineolytica LLG109, - transforming suitable host cells with said vectors, - culturing the host cells under suitable conditions to express any enzyme of interest, - screening for positive clones by determining any 0-1,3- glucanase activity of the enzyme produced by such clones, - selection of clones, and - isolating the enzyme encoding DNA from such clones. The general method is further disclosed in WO 93/11249 which is hereby incorporated by reference. A more detailed description of the method is given below. The DNA sequence coding for the enzyme may for instance be isolated from Oerskovia xanthineolytica strain LLG109 (Lechevalier, Int. J. Sys. Bacteriol., 22(4), p. 260, 1972), and selecting for clones expressing the appropriate enzyme activity (i.e. 0-1,3-glucanase activity as defined by the ability of the enzyme to hydrolyse 0-1,3-glucan bonds of a suitable substrate such as laminarin or AZCL-curdlan, cf. the Materials and Methods section hereinafter) . The Oerskovia xanthineolytica LLG109 strain has been deposited by the inventors according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the Deutshe Sam lung von Mikroorganismen und Zellkulturen GmbH. , (DSM) . Deposit date : 13.10.95 Depositor's ref.: NN049107 (Oerskovia xanthineolytica LLG109) DSM designation : Oerskovia xanthineolytica (or Cellulomonas cellulans) DSM No. 10297 Being an International Depository Authority under the Budapest Treaty, Deutshe Sammlung von Mikroorganismen und Zellkulturen GmbH. , affords permanence of the deposit in accordance with the rules and regulations of said treaty, vide in particular Rule 9. Access to the deposit will be available during the pendency of this patent application to one determined by the Commis¬ sioner of the United States Patent and Trademark Office to be entitled thereto under 37 C.F.R. Par. 1.14 and 35 U.S.C. Par. 122. Also, the above mentioned deposits fulfil the requirements of European patent applications relating to micro-organisms according to Rule 28 EPC. The above mentioned deposit represent substantially pure cultures of the isolated bacteria. The deposit is available as required by foreign patent laws in countries wherein counter¬ parts of the subject application, or its progeny are filed. However, it should be understood that the availability of the deposited strains does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. The appropriate DNA sequence may then be isolated from the clone by standard procedures, e .g. as described in Materials and Methods section. According to the invention the DNA construct comprises a DNA sequence, which a) comprises the DNA sequence shown in SEQ ID No. 1, or b) comprises an analogue of the DNA sequence shown in SEQ ID No. 1 , which i) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1, and ii) 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 iii) encodes a polypeptide which is immunologically reactive with an antibody raised against a purified 0-1,3-glucanase encoded by the DNA sequence shown in SEQ ID No. 1 derived from OersJtovia xanthineolytica LLG109. In an embodiment of the invention the DNA construct comprises the sequence shown in SEQ ID No. 1 or SEQ ID No. 3. The polypeptide encoded by said DNA sequences shown in SEQ ID No. 1 and SEQ ID No. 3 are shown in SEQ ID No. 2 and SEQ ID No. 3. A preferred method of amplifying specific DNA sequences are by the use of polymerase chain reaction (PCR) using degenerate oligonucleotide probes synthesized. For instance, the PCR may be carried out using the techniques described in US Patent No. 4,683,202 or by R.K. Saiki et al. (1988), Science, 239, 487-491 It is expected that a DNA sequence coding for a homologous enzyme, i.e. an analogous DNA sequence, is obtainable from other microorganisms. For instance, the DNA sequence may be derived from another microorganism, in particular either a fungus or bacteria. Such DNA sequences may originates from fungi, comprising a strain of an Aspergillus sp. , in particular a strain of A. aσuleatus or A. niger, a strain of Trichoderma sp. , in particu¬ lar a strain of T. reesie , T. viride , T. longibrachiatum or T. koningii , T. harzianum or a strain of a Fusarium sp. , in particular a strain of F. oxysporum, or a strain of a Humicola sp. . Further a DNA sequence encoding a homologous enzyme may be expected to derive from bacteria, such as another strain of a Oerskovia sp. , or a strain of an Arthrobacter sp. , Cytophaga sp. , --hodoti-er-nus sp. , in particular a strain of Rh . marinuε, or a strain of a Clostrium , in particular strains of Cl . thermocellum, or a strain of Bacillus sp. , in particular strains of B . lichenitormiε ., B . amyloliquefaciens, or B. circulans . Alternatively, the DNA coding for a 0-1,3-glucanase of the invention may, in accordance with well-known procedures, conveniently be isolated from DNA from 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 a partial nucleotide sequence of the sequence shown in SEQ ID No. 1. The DNA sequence may subsequently be inserted into a recombi¬ nant 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 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 vector, the DNA sequence encoding the 0-1,3-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 either homologous or heterologous to the host cell. It is preferred to use a vector under control of the promoter for the maltogenic 0-1,3-amylase from Bacillus stearothermophilus and/or the signal of Bacillus stearothermophilus . In a specific embodiment the expression vector comprises the plasmid pPFFl shown in Figure 1. The procedures used to ligate the DNA sequences coding for the 0-1,3-glucanase, 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., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY, 1989) . The host cell which is transformed with the DNA sequence encoding the enzyme of the invention may be either eukaryotic cells or prokaryotic cells. Suitable prokaryotic host cells are in particular bacterial cells. Examples of such bacterial host cells which, on cultivation, are capable of producing the novel enzyme of the invention are gram-positive bacteria such as strains of Bacillus , such as strains of B . subtilis, B . licheniformis, B . lentus, B . brevis, B . stearothermophilus, B . alkalophilus , B . amyloliquefaciens, B . coagulanε, B . circulans, B . lautus, B . megaterium or B . thuringiensis, or strains of Streptomyces, such as S . lividans or S. murinus , or gram-negative bacteria such as Escherichia coli . The transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra , 1989). When expressing the enzyme in bacteria such as E. coli , the polypeptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies) , or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In the latter case, the polypeptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the polypeptide. In a specific embodiments of the invention the bacterial host cell is a strain of Bacillus subtilis, especially the strain B . subtilis DN1885 or the protease deficient strain B. suJtilis TOC46. Suitable eukaryotic cells are in particular fungal cells such as yeasts or filamentous fungal cells. Examples of suitable yeast cells include cells of Saccharomyces spp. , in particular strains of Saccharomyces cerevisiae , Saccharomyces kluyveri, Sacchromyces uvarum , or Schizosaccharo yces spp., such as Schizosaccharomyces pombe . Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides there from are described, e.g. in US patent no. 4,599,311, US patent no. 4,931,373, US patent no. 4,870,008, US patent no. 5,037,743, and US patent no. 4,845,075, all of which are hereby incorporated by refer- ence. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e .g. leucine. A preferred vector for use in yeast is the P0T1 vector dis¬ closed in US patent no. 4,931,373. The DNA sequence encoding the polypeptide of the invention may be preceded by a signal sequence and optionally a leader sequence , e .g. as described above. Further examples of suitable yeast cells are strains of Kluyvero yces spp., such as K. lactis , or Hansenula spp., e . g. H . polymorpha , or Pichia spp., e .g. P. pastoriε, Yarrowia spp., such as Yarrowia lipolytica (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US patent no. 4,882,279). Examples of other fungal cells are cells of filamentous fungi, e . g. Aspergillus spp., Neuroεpora spp., Fuεarium spp. or Trichoderma spp., in particular strains of A. oryzae , A. nidulanε or A. niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277, EP 238 023 and EP 184 438. The transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., Gene 78, p. 147-156, 1989. When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct of the invention, conveni¬ ently by integrating the DNA construct in the host chromosome to obtain a recombinant host cell. This integration is gen¬ erally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e .g. by homologous or heterologous recombination. 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 cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection) . The expressed 0-1,3-glucanase produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt. e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question. In a still further aspect, the present invention relates to a novel enzyme preparation useful for the modification or degradation of 0-glucan containing materials, said preparation being enriched in an enzyme exhibiting 0-1,3-glucanase activity as described above. The enzyme preparation having been enriched with the novel 0- 1,3-glucanase enzyme of the invention may e . g. be an enzyme preparation comprising multiple enzymatic activities, in particular an enzyme preparation comprising different enzyme activities required for the modification or degradation of microbial cell walls. Examples of such enzyme preparations include lytic enzyme systems, in particular of microbial (fungal or bacterial) origin, e .g. derived from a strain of Trichoderma , such as Trichoderma harzianum, Trichoderma viride or Trichoderma reesie , a strain of Oerskovia sp. , such as Oerskovia xanthineolytica (sometimes called Cellulomonas cellelanε) , a strain of ArtήroJaσter sp. such as Art roJaσter luteuε, a strain of Rhizoctonia sp. or Cytophaga sp. , a strain of a Staphylococcuε sp. , or a strain of Streptomyceε sp.. Commercially available enzyme preparations which may conveni- ently be boosted with an enzyme of the invention includes Novozyme® 234 and Cereflo 200L, both available from Novo Nordisk A/S, Denmark, Cellulase (available from Merck) , Cellulase CP and Cellulase CT (both available from Sturge) , and/or Chitinase (available from Sigma) , Zymolase from Kirin Breweries. In the present context, the term "enriched" is intended to indicate that the 0-1,3-glucanase 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 0-1,3,-glucanase activity may be one which comprises an enzyme of the invention as the major enzymatic component, e .g. a mono-component enzyme composition. 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 a 0-1,3-glucanase of the invention, contain one or more other cell wall degrading enzymes, for instance those with celluly- tic, mannanolytic, chitinolytic or proteolytic activities such as endo- and exo-glucanase, mannanase, endo- and exo- chitinase, protease, or - or 0-mannosidase. The additional enzyme(s) may be producible by means of a microorganism belonging to the genus Aspergillus , preferably Aspergillus niger, Aspergillus aculeatuε , Aspergilluε aw amor i or Aspergil¬ lus oryzae , or the genus Trichoderma , or the genus Oerskovia , the genus Cytophaga , or the genus Arthrobacter or any of the microorganisms mentioned above in connection with the commer¬ cially available enzyme preparations. Examples are given below of preferred uses of the enzyme preparation of the invention. The dosage of the enzyme composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art. The enzyme preparation according to the invention is preferably used as an agent for degradation or modification of 0-glucan containing material such as microbial cell walls. In particu¬ lar, 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 composi¬ tion 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 0-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 transform¬ ation. Further, the enzyme or enzyme preparation according to the 5 invention may be used in the preparation of pharmaceuticals, especially products entrapped inside the cells in the cyto¬ plasmic membrane, the periplasmic space and/or the cell wall. In addition, the enzyme preparation of the invention may be 10 used in the modification of 0-glucans such as curdlan and laminarin. The invention is described in further detail in the following examples which are not in any way intended to limit the scope 15 of the invention as claimed. METHODS AND MATERIALS 20 Donor organism: Oerskovia xanthineolytica LLG109 (M. Lechevalier, Int. J. sys. Bacteriol, 22(4) , p. 260, 1972). Oerskovia xanthineolytica 73/14 (Shen et al., J. Biol. Chem, 25266, p. 1058, 1991) . Deposited microorganisms: Oerskovia xanthineolytica LLG109 strain has been deposited at 30 the Deutshe Sammlung von Mikroorganismen und Zellkulturen GmbH., (DSM). Deposit date : 13.10.95 Depositor's ref.: NN049107 (Oerskovia xanthineolytica LLG109) 35 DSM designation : Oerskovia xanthineolytica (or Cellulomonas cellulans) DSM No. 10297 Host organism: Bacillus subtiliε DN1885 (Diderichsen et al., Journal of Bacteriology, vol. 172, p. 4315-4321, 1990) Bacillus subtiliε protease deficient strain ToC46 (Diderichen et al., supra, 1990). J-T.coli JM109 (Yanish-Perron et al., Gene 33, p. 103-199, 1985) PF8A: E. coli JM109 harbouring plasmid pPF8A PF15BK: B. subtilis DN1885 harbouring plasmid pPF15BK PFllBgK: B . subtilis DN1885 harbouring plasmid pPFUBgK PFF1: B. subtilis DN1885 harbouring plasmid pPFFl Primers: DS96: 5' cggaattccatgcctcacgacaggaggaa 3' DS97: 5' tatggatccagatctcgtcgaagcacgtg 3' Primers synthesized for the 5' region of the yeast lytic 57 KDa 0-1,3-glucanase from O. xanthineolytica 73/14. DS121: 5' ccN ggN gaY YtN YtN tgg 3' DS141: 3' atR gtY ggN gtY atR cc 5' DS140: 5' ttY gtN gaY ggN caR caR tt 3' DS142: 3' aaR caN ctR ccN gtY gtY aa 5' DS143: 3' ctR atR caN KcN caN atR ct 5' Y = C or T R = A or T K = G or T N - C, T, A or G Synthesized according to known amino acid sequences of the 0- 1,3-glucanase (Ventom and Asenjo, supra, 1991). DK15: 5' cgtaσtcσσσggtggcttcagcgαcgtttggattgtac 3' ======================.=== Aval DK16: 5' gatgcaagcttgcattacgaaaggagac 3' =-=__.__.__.=_==__=---===-=====-= Hindlll Primers used for plasmid pDN1777 DNA amplification by PCR. Double underlined nucleotide sequences correspond to B . εtearothermophiluε, whereas the underlined nucleotide sequence correspond to O . Xanthineolytica LLG109 5 Plas ids: pPFFl: See figure 1 pPF8A: 2.7 Kb BamHI-BamHI fragment from O . xanthineolytica LLG109 (see figure 2) . pDN520: (Diderichsen, B. and Christiansen, L. , FEMS Microb- 10 iology Letters 56, p. 53-60, 1988) . pUC18: (Yanish-Perron, C, Viera, J and Messing, J. , Gene, 33, p. 103-199, 1985) pDN2801: (Diderichsen, B. , Wested, U. , Hedegaard, L. , Jensen, B.R. Sjøholm, C. , Journal of Bacteriology, >fl, 15172, p. 4315-4321, 1990) Promoter: Maltogenic α-amylase from Bacilluε s tear other ophilus (Diderichen et al., supra , 1988) 20 METHODS: Isolation of genomic DNA: Chromosomal DNA from O . xanthineolytica was prepared according to Meade et al., J. 25 Bacteriol., 149(1), p. 114-122, 1982) Construct of a genomic DNA partial library for O. xanthineolytica LLG109 Chromosomal DNA from O . xanthineolytica LLG109 was partially 30 digested with BamHI. The digested DNA was fractionated by agarose gel electrophoresis. Fragments with sizes of about 1.6 to 4 kb were isolated and purified. The resulting BamHI fragments were mixed with BamHI-digested-Bacterial Alkaline dephosphorilated pUC18 plasmid vector (pUC18 BamHI/BAP from 35 Pharmacia) , and ligate with T4 ligase (BRL) . This ligation mixture was used to transform electrocompetent E. coli JM109 cells by electroporation (Bio-Rad Gene Pulser; 125 μFD capaci¬ tance, 200Ω Resistance) . E coli JM109 cells were made competent according to Sambrook et al., "A laboratory manual, Cold Spring Harbor Laboratory Press, New York,, 1989. Screening of the partial DNA library from O. xanthineolytica 5 LLG109 with radiolabelled DS140/DS143 PCR product: The partial DNA library from strain LLG109 was plated in a total of 11 plates (with about 50 white (recombinant) colonies/plate) . The library was replicated on sterile nylon membranes (Hybond-N, Amersham) as described by Sambrook, supra , 101989) . The replica membranes were probed with the radiolabelled PCR product according to Sambrook et al., supra , 1989, under the following conditions: Hybridization temperature: 65°C, hybridization solution (100 ml) 6XSSC, 0.5% non-fat dried milk. Hybridization was carried out overnight (18 hours) . After 15 hybridization, filters were washed as follows: 10 min + 15 min in 200 ml of 2x SSC at 65°C and 5 min at 65°C in 0.2x SSC, 0.1% SDS. Filters were subjected to autoradiography (Fuji Film) overnight as -70°C to detect 32 P-labelled probe on filter. 20 PCR (Polymerase Chain Reaction) Amplification: PCR amplification from O . xanthineolytica LLG109 The PCR reactions were carried out using 100 pmols of each degenerate primer per reaction. An amount of 100 ng of Oerskovia DNA was used per reaction. Taq DNA Polymerase from 25 Promega was used using the conditions recommended by the manufacturer. Cycling conditions were as followed: Hot start: 95°C for 1 minute 40 cycles. The PCR product was cloned in pUClβ/Smal as described by 30 Kanungo and Pandey, Biotechniques, 14(6), p. 912, 1993, for subsequent sequencing. Gel electrophoresis of DNA and Southern Blotting Agarose gel electrophoresis, and Southern Blotting, was used to 35 analyze plasmids, restriction endonuclease fragments, ligation products, PCR reaction, according to Sambrook et al., supra , 1989. Recovery of DNA from agarose gels was done according to Sambrook et al., supra , 1989. Radiolabelling of double-stranded DNA fragments Radiolabelling of double-stranded DNA fragments was done according to Sambrook et al., supra , 1989. DNA hybridization experiments Hybridization of Southern blots on nylon filters (Hybond-N, Amersham) with 32 P-labelled PCR probe were carried out following methods described by Sambrook et al., supra , 1989. The hybrid¬ ization conditions were as follows: Hybridization temperature: 65°C. Hybridization solution: 6xSSC, 0,5% non-fat dried milk). The hybridization solution was carried out overnight. Transformation of E. coli i Cells of E. coli were transformed by electroporation as describ¬ ed in the manual for the BIO-RAD Gene Pulser electroporation apparatus. Transformation of B . subtili i Competent cells were prepared and transformed as described by Yasbin, R.E., Wilson, G.A. and Young, F.E., J. Bacteriol. vol. 121, p. 296-304, 1975. Identification of positive clones: The identification was done according to Sambrook et al., εupra , 1989. DNA sequencing DNA sequencing was done using the same SEQUENASE sequencing kit from USBI following the manufactures recommendations. DNA compressions were resolved by using the reagent kit for DNA sequencing using 7-deaza-dGTP and sequenase (also from USBI) . Isolation of plasmid DNA: E. coli : The isolation of E. coli plasmid was performed according to Sambrook et al., εupra , 1989, using the method described in Promega Protocols and Application Guide) B . subtiliε : The isolation of B . εubtiliε was done as described by Kieser, T. , Plasmid, 12, p. 19-36, 1984. Immunological cross-reactivity: Antibodies to be used in determining immunological cross-reactivity may be prepared by use of a purified 0-1,3,-glucanase. More specifically, antiserum against the 0-1,3-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, Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically p. 27-31) . Purified immunoglobulins may be obtained from the antisera, for example by salt precipi¬ tation ((NH 4 ) 2 S0 4 ) , followed by dialysis and ion exchange chromatography, e . g . on DEAE-Sephadex. Immunochemical charac¬ terization 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., εupra , Chapters 3 and 4) , or by rocket immunoelectrophoresis (N. Axelsen et al.. Chapter 2) . Media LB aαar Bacto-tryptone lOg/1 Bacto yest extract 5g/l NaCl I0g/1 Bacto agar 15g/l adjusted to pH 7.5 with NaOH All autoclaved at 121°C, 30 minutes. LBGP agar LB agar chanting 10 mM potassium phosphate pH 7.0, 0.4 % glucose All autoclaved at 121°C for 30 minutes. Jacm-7-medium: Maltodextrin 02 40 gram (average chain length=12) Sucrose MgS0 4 , 7H 2 0 KH 2 P0 4 Na 2 HP0 4 , 2H 2 0 Mikrosoy Tapwater at pH adjusted to 7.0 with NaOH The nitrogen sources, Casitone and (NH 4 ) 2 HP0 4 were added separately after autoclaving as concentrated solutions, the final concentrations were: Casitone 9.6 gram/1 (NH 4 ) 2 HP0 4 10.8 gram/1 All autoclaved at 121°C for 60 minutes. Fermentation broth-1: Casitone 152 gram KH 2 P0 4 5.16 gram Na 2 HP0 4 ,2H 2 0 5.12 gram K 2 S0 4 1.09 gram MgClj,6H 2 0 1.11 gram Lic-tracemetals 20 ml Antifoam SB2121 1 ml (Struktol SB2121 from Schill & Seilacher GmbH, Hamburg, Germany) The volume was adjusted to 1.0 litre with tap water. The pH was adjusted to 5.0 with H 3 P0 4 before sterilization in an autoclave at 121 β C for 60 minutes. Sucrose feed solution: Content in 1 litre flask: MgCl 2 ,6H 2 0 8.08 gram Lic-tracemetals 34 ml Antifoam SB2121 1.3 ml Sucrose 241 gram Tapwater to 500 ml Also autoclaved at 121°C for 60 minutes, CAL 18-2 medium: Yeast extract 40.0 gram MgS0 4 1.3 gram Maltodextrin 02 50.0 gram NaH 2 P0 4 20.0 gram Trace metals 1 6.67 ml Deionized water to 1 litre The pH was adjusted to 6.0 with NaOH and autoclaved at 121°C for 60 minutes. TRACE METAL SOLUTIONS Mikrosoy: rog/1 MnS0 4 ,H 2 0 500 FeS0 4 ,7H 2 0 2000 CUS0 4 ,5H 2 0 200 ZnCl 2 200 Na 2 B 4 O 7 ,10H 2 O 1000 BaCl 2 ,2H 2 0 100 (NH 4 ) 6 Mθ 7 0 24 , 4H 2 0 100 Na 3 -citrat,H 2 0 10000 All autoclaved at 121°C for 60 minutes. Mikrosov: mg/1 MnS0 4 ,H 2 0 500 FeS0 4 ,7H 2 0 2000 CuS0 4 ,5H 2 0 200 ZnCl 2 200 Na 2 B 4 O 7 ,10H 2 O 1000 BaCl 2 ,2H 2 0 100 (NH 4 ) ^0,0 24 ,4^0 100 Na 3 -citrat,H 2 0 10000 All autoclaved at 121°C for 60 minutes Trace metals 1: MnS0 4 4.48 g FeCl 3 ,6H20 3.33 g CuS0 4 ,5H 2 0 0.625 g ZnS0 4 ,7H 2 0 7.12 g Na 2 Mo0 4 2.0 g KI 1.0 g Deionized water to 1 litre 500 All autoclaved at 121°C for 60 minutes. Enzymes: Native 0-1,3-glucanase from Oerεkovia xanthineolytica LLG109 (Ventom and Asenjo, Enzyme Microb. Technol., 13, p. 71 , 1991) Substrate: AZCL-curdlan: Megazyme, Sydney, Australia Laminarin: Sigma Enzyme assay: Assay conditions: Shaking waterbath, 37°C for 1 hour. Incubation mixture: 0.5 ml supernatant + 0.5 ml 1 w/v% AZCL- curdlan + 0.5 ml acetate-buffer (pH 4.0) . Curdlan was also dis¬ solved 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. EXAMPLES Example l PCR amplification of native genomic DNA from O . xanthineolytica LLG109: Native genomic DNA from O . xanthineolytica was prepared as described above and amplified using PCR technic. Primers DS96 and DS97 were synthesized for the 5' region of the yeast-lytic 57 kDa.0-1,3-glucanase gene from O . xanthineolytica LLG109 starting from the putative start codon and for the 3' region just downstream from the putative stop codon. Bases for sequences for EcoRI and BamHI were introduced at the 5' primer (DS96) and 3' primer (DS97) . PCR amplification from genomic DNA from strain O . xanthineolytica LLG109 using these primers gave a 1.7 kb DNA fragment. The PCR-fragment was checked by digesting it at various restriction sites internal to the gene. Example 2 Partial amino acid sequence determination from the 26 kDa lvtic fi-1.3-glucanase from O. xanthineolytica LLG109. The following amino acid sequences were initially obtained from the N-terminal of the native 0-1,3-glucanase and from peptides generated by trypsin digestion of the native enzyme. Proteolytic digestion, peptide separation and amino acid sequencing were done according to standard procedures. N-Terminal native enzyme: N-Ala-Pro-Gly-Asp-Leu-Leu-Trp-Xaa-Asp-Glu-Phe- Peptide 1: Tyr-Gln-Pro-Gln-Tyr-Gly-Arg-Ile-Glu-Ala—Arg-Ile-Gln-Pro-Arg- Gly- Peptide 2: Phe-Val-Asp-Gly-Gln-Gln-Phe-Xaa-Arg-Val- Peptide 3: Val-Asp-Tyr-Val-Arg-Val-Tyr-Asp- Example 3 O . xanthineolytica LLG109 chromosomal DNA amplification by PCR. Chromosomal DNA from O. xanthineolytica LLG109 was amplified by PCR using different combinations of the mixed oligonucleotides synthesized on the basis of reverse translation of amino acid sequence from 0-1,3-glucanase. PCR reaction using primer DS140 (reverse sense translation of the amino acid sequence FVDGQQF from peptide 2) and DS143 (reverse antisense translation of the amino acid sequence DYVRVY from peptide 3) produced a 180 bp DNA fragment. This PCR product was subsequently cloned into pUC18 and transformed into E. coli JM109 in order to determine its complete nucleotide sequence (see SEQ ID No. 3) . Example 4 Southern blot hybridization analysis of chromosomal DNA from strain LLG109 using DS140/DS143 PCR product as a probe. Chromosomal DNA from strain LLG109 was analyzed by Southern blotting using the radiolabelled DS140/DS143 PCR product as a probe under stringent conditions. A strong positive signal from BamHI-BamHI band of approximately 2.65 kb or from an about 8 kb Kpnl-Kpnl band or a 1.5 Kb BamHI-Kpnl band. In contrast, the PCR probe strongly hybridised (under the same conditions) to either a 8 kb BamHI-BamHI band, a 8 kb Kpnl-Kpnl band or an about 5 kb BamHI-Kpnl band in Southern blot hybrid¬ ization analysis of chromosomal DNA from Strain 73/14. In both cases, the pattern of bands, in the Southern blots from both Oerεkovia strains showing strong hybridization to the radiolabelled probe, was different to that observed using the PCR product DS133/DS135. Therefore, it was concluded that both O . xanthineolytica strain LLG109 and 73/14 probably had at least two different 0-1,3- glucanase genes. Example 5 Cloning of 26 kDa 0-1.3-glucanase gene from O . xanthineolytica LLG109 in E. coli In order to clone the 2.7 kb BamHI-fragment hybridizing to the DS140/DS143 PCR-probe, appropriate sized (2.7 kb +/- 0.5 kb) BamHI DNA-fragments from the chromosome of strain LLG109 were purified and ligated to the BamHI site of pUC18. E . coli JM109 was transformed by electroporation with the ligates and screened by colony hybridization using the same DS140/143 PCR- probe and conditions mentioned above. A clone from the partial gene bank harbouring recombinant plasmid pPF8A was isolated. This plasmid was mapped by restric¬ tion enzyme analysis. Location of the region of the plasmid homologous to the DS140/DS143 PCR-probe and insert orientation was determined by Southern blotting. Plasmid pPF8A contained 2.7 kb BamHI insert. In the plasmid the probe was hybridizing to the 1.5 kb BamHI- Kpnl fragment contained in the 2.7 kb BamHI insert. This fact was consistent with the genomic Southern blot hybridization result using DS140/DS143 PCR probe. Example 6 Nucleotide sequence of the 0-1.3-glucanase gene The nucleotide sequence of the 1.5 kb BamHI-Kpnl fragment from pPF8A was determined by the Dideoxi Chain Termination method. Double-stranded DNA was denaturated by alkali or heat treatment before using it as a template for sequencing reactions. Both strands of this region from pPF8A were sequences according to the sequence strategy shown. The complete nucleotide sequence of the 1.5 kb BamHI-Kpnl fragment and the deduced amino acid sequence are shown in SEQ ID No. 1 below. The nucleotide sequence contains an Open Reading Frame (ORF) of 921 nucleotides encoding a protein composed of 306 amino acids. Amino acid sequences identical or very similar to those existing in the native protein were present along the amino acid sequence predicted from the DNA sequence (amino acid residues different are discussed below) . The codon ATG is used as initiation codon in this OFR. A putative Ribosome Binding Site (RBS) is present 4 nucleotides upstream the initiation codon. Upstream of this ORF, there are two other in-frame ATG codons (nucleotides 321 and 351) . However, it is unlikely that any of these functions as an initiation codon, because none of them are preceded by putative RBS's in the expected positions. Computer scanning of the sequence upstream of the coding region could not find sequences compatible with an E . coli type of promoter. In the 3'-flanking region, which is downstream the termination codon TGA, there is a G+C-rich region (69% C+G) 13 base pairs inverted repeat sequence. This sequence can make a stem-loop structure and possibly may function as a transcrip¬ tion termination signal. The 1.5 kb BamHI-Kpnl DNA fragment has a G+C content of 67%, and the G+C content of the ORF was, similar to that of Oerεkovia chromosomal DNA reported previous¬ ly (70-75% G+C) (Stackebrandt and Prauser, System. Appl. Microbiol., 14, p. 261-265, 1991) . Amino acid sequence of the precursor of the β-1.3 glucanase The ORF of the 0-1,3 glucanase gene encode a 306 amino acid protein. The NH 2 -terminal end of the deduced amino acid sequence exhibits signal sequence characteristics found in other secreted proteins (von Heine, Nucl. Acids Res., 14(11), p. 4683, 1986). Following the initiation codon there are three positive charged amino acid residues (histidine 5 and arginine 12 and 13) which are trailed by a long stretch of hydrophobic amino acid (Leu ,7 -Ala 23 and Ala 26 -Ala 35 ) . The computer prediction for prokaryotic secretory signal sequence gave the following positions as best potential cleavage sites: i) potential cleavage site between position 35 and 36. The NH 2 -terminal sequence of the native mature form of 0-1,3-glucanase purified by Ventom and Asenjo, supra, 1991, from strain LLG109 fermenta¬ tion broth was determined as APGLLWSDEFDGAAGS. This amino acid sequence shows a high similarity to that predicted from the nucleotide sequence in positions Thr^-Ser 80 : TPESLAWSDEFDGAAGS♦ Amino acid differences (see figure 3) observed between the predicted amino acid sequence and the sequence obtained from the native 0-1,3-glucanase (underlined amino acid residues) suggest that the ORF found in pPF8A may code for a different 0- 1,3-glucanase, an isoenzyme to the enzyme described by Ventom & Asenjo, supra , 1991. As a result, the amino acid residue Thr 64 forms a NH 2 -terminal end of the 0-1,3-glucanase isoenzyme. Hence, the peptide containing 63 amino acids (Met'-Val 63 ) should be considered as a prepro-region. The molecular weight of the mature 0-glucanase (Thr^-Gln 243 ) was calculated to be 26.278, which is similar to that previously calculated from SDS-PAGE by Ventom and Asenjo, εupra , 1991, for the characterized 0-1,3- glucanase. The predicted pi for this new enzyme is 3.77, which is so much lower than the previously reported (Ventom and Asenjo, supra , 1991) pi of 5.0, that no doubts can be left that a new hitherto unknown 0-glucanase was found. Example 7 Cloning and expression of the 26 kDa 0-1.3-glucanase gene in Bacillus subtiliε The 1.5 kb BamHI-Kpnl fragment from pPF8A was cloned in B . subtilis DN1885 on plasmid pDN2801 under the control of the promoter for the maltogenic α-amylase from B . stearotherophiluε (Diderichsen, εupra , 1990) producing the plasmid pPF15BK (figure 4) . The 1.1 kb Bglll-Kpnl fragment contained in the 1.5 kb BamHI-Kpnl fragment was cloned in the same way to give pPFUBgK (figure 5) . However, neither plasmid transformed (Yasbin et al., J. Bacteriol. 12, p. 296-304, 1975) into B . εubtiliε DN1885 gave rise to any detectable 0-1,3-glucanase activity on LBGP agar plates containing AZCL-curdlan nor in liquid culture (CAL 18-2, 3 days, 30°C, 250 rpm) supernatants. Alternatively, expression of the 0-glucanase gene was performed by replacing the native expression signals with those from the well-expressed Bacilluε stearothermophilus gene mentioned above. A perfect fusion of the nucleotide sequence for the promoter, RBS and signal peptide of the maltogenic α-amylase gene from B . εtearothermophiluε and the codons for Thr64 and the rest of the 0-1,3-glucanase ORF, including the terminator, was made. This final construct pPFFl was made as follows: A 0.2 kb Hindlll-Aval fragment from pPFUBgK was replaced by a 0.15 kb Hindlll-Aval fragment made by PCR. The PCR fragment con¬ tained the RBS and the signalpeptide coding regions of the B.stearothermophilus maltogenic α-amylase. The PCR fragment was obtained following pDN520 (Diderichsen et al., supra, 1988) DNA amplification using primers DK15 and DK16. Plasmid pPFFl was finally transformed into B.subtilis DN1885 (Yasbin et al, εupra , 1975). In this case 0-1,3-glucanase activity could be detected after 24 hours, 37°C on laminarin (0.04%) containing 5 LB agar plates after staining with congo red. Finally pPFFl was transformed into the protease deficient host B. subtilis ToC46. Example 8 10 Fermentation and production of 0-1.3-glucanase from Bacillus subtiliε DN1885/PPFF1 The strain Bacilluε subtilis DN1885/pPFFl was grown in a 2 litre fermentation vessel supplied with a magnetic coupled 15 stirrer drive, pH- and temperature control and a peristaltic pump to add the carbon source at fixed rates. 100 μl of the - 80°C stock culture of strain DN1885/pPFFl was used to inoculate a 500 ml shakeflask containing 100 ml of Jacm-7-broth and 1 mg of chloramphenicol and propagated for 24 hours at 250 rpm and 2030°C on a rotating table. This culture (pH=7.3 and 00 600 =22) was used to inoculate the fermenter already containing 1.0 litre Fermentation broth 1. Biomass growth and enzyme formation 25 Before inoculation of the culture as described above, 32 gram of the Sucrose solution was added to the fermenter. Also steri¬ le filtered air was sparged into the fermenter at the bottom drive at a rate of 1 litre/minute. The stirrer speed was at the beginning set to 500 rpm, but automatically coupled to the dis- 30 solved oxygen tension signal at a set point of 20% DOT (Dis¬ solved Oxygen Tension) . A peristaltic Watson-Marlow pump was used to feed the Sucrose feed solution with a constant rate of 7.1 gram/hour after 10 hours of growth, this forced the stirrer to run close to the maximum value of 1100 rpm and brought the 35 DOT close to 0. After 19 hours we therefore reduced the sucrose feed rate to -6.0 gram/hour, which was kept until the bottle was emptied after -82 hours fermentation time. The fermentation was stopped after 93 hours, cooled down to 4'C and stored for later recovery. The temperature was controlled at 34.0°C +/- 0.1°C, pH to 6.00 +/- 0.10 by addition of diluted ammonia in water or 5 v/v% H 3 P0 4 . Samples were taking daily and pH, dry weight biomass and 0-1,3- glucanase activity determined as described in the section Materials and Methods. Enzyme concentrations in the culture su- pernatants were determined by AZCL-curdlan hydrolysis. Fermentation results shown in Table 1. TABLE 1 Example 9 Growth and enzvme expression on CAL 18-2 medium. The strain B . subtilis DN1885/pPFFl was grown on LB agar plates containing 10 μg/ml Chloramphenicol for 3 days at 30°C. A single colony was transferred to a 500 ml shakeflask containing 100 ml of CAL 18-2 medium and 1 mg of chloramphenicol. Growth was propagated at 30°C, on a rotating table at 250 RPM, for 3 days. Samples were taken every day and activity, pH, ODβoo and dry weight biomass determined. The activity determination was made as described in the section Methods and Materials, except that we used 0.05 ml supernatant + 0.25 ml curdlan + 0.2 ml water and stopped with 2 ml of stop reagent. Also the incuba- tion time was extended to 20 hours. Results are displayed in Table 2. TABLE 2 nd= not -dBπnin-d Example 10 N-terminal amino acid seguence determination and mass spec- trometry of purified recombinant 0-1.3-glucanase. The fermentation broth containing the recombinant 0-1,3-gluca- nase was ultrafiltrated and washed with 20 mM Tris-HCl pH 8.5 in a Filtron concentrator equipped with a Minisette 10 kDa membrane. The sample was applied on a Q-Sepharose column equilibrated with 20 mM Tris-HCl pH 8.5 and the 0-1,3-glucanase was eluted with a linear gradient from 0 to 1 M NaCl in 10 column volumes. The fractions containing 0-1,3-glucanase activity were pooled. The pool was made 1.7 M with respect to ammonium sulfate and pH was adjusted to 6.5. The pool was further purified on a Phenyl-Sepharose column equilibrated with 30 mM MOPS containing 1.7 M ammonium sulfate pH 6.5. Elution of the 0-1,3-glucanase was performed with 10 mM Tris-HCl pH 8.5 using a linear gradient in 10 column volumes. The fractions containing 0-1,3-glucanase activity were pooled and dialyzed extensively against 10 mM sodium borate pH 9.0. The dialyzed sample was applied on a Mono Q column equilibrated with 10 mM sodium borate and the 0-1,3-glucanase was eluted with a linear gradient from 0 to 1 M NaCl in 40 column volumes. The N-terminal amino acid sequence of the purified recombinant 0-1,3-glucanase is identical to the N-terminal sequence deduced from the SEQ ID No. 1. Matrix assisted laser desorption ionisation time-of-flight mass spectrometry of the purified recombinant 0-1,3-glucanase gave a mass of 26 462 Da which within the experimental error is in accordance with the mass calculated from the amino acid sequen¬ ce. ABBREVIATIONS AMINO ACIDS SEQUENCE LISTING (1) GENERAL __NFC-FMATICN: (i) APPLICANT: (A) NAME: Novo Nordisk A S (B) SU EET: Nσvo Alle (C) CITY: Bagsvaerd (E) CDUNIRY: Deπnark (F) POSTAL GODE (ZIP) : EK-2880 (G) TET-EfH-WE: 445 4444 8888 (H) TELEFAX: +45 4444 3256 (ii) TTTI_E OF --NV--NTION: Novel enzyme with beta-l,3-glucar--_se activity (iii) NUMBER OF SEQUENCES: 3 (iv) CCMPUTER READABI-E PCPM: (A) MEDIUM TYPE: Floppy disk (B) CCMFUT--R: HEM PC cααpatible (C) CP__RKΠNG SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Paten m Release #1.0, Version #1.30B (IPO) (2) --NFQFMATIC_N PCR SEQ ID NO: 1: (i) SEQUENCE CHARACTE- I--TICS: (A) I--H-TH: 1459 base pairs (B) TYPE: nucleic acid (C) SΩttNDEΣNESS: single (D) TOPC8-OGY: linear (ii) MXECULE TYPE: DNA (ger-a- c) (iii) HYPOIHEriCAL: O (iv) ANΠ-SENSE: NO (vi) ORIGINAL SOURCE: (B) SU AIN: Oerskovia xanthineolytica L--G109 (ix) FEATURE: (ix) FEATURE: (A) NAME/KEY: CDS (B) KX-A-nCN:353..1270 (xi) SEQUENCE D-ϊSCRIPri-N: SEQ ID NO: 1: GGAIOa-GIG -YK-O∞GAAA OCTK-GICAT GAGGGfTK-GG IGCGCTIX-AG AOX-CCCCGC 60 GCK-TCX---AC O-CIGC- GAG GAO3G03CAC AOX-TOCGGT GC-V-X-A TCG GG-V-GTCQGG 120 αr-¥-GAGC-T GGTOQGCATC -ttA-K-AOGGC AKX-TTOQGA TGACACTCIT e-A_JO-. l.-lAG 180 C0QGGGC3-AC AA-X GIGGT U-llU iU-ti AGGAGCTOCT CGCSV30CCTC GTOTCAGAGG 240 IGOOCGCTTC GACGCO-GCC CA----»GE__AC GGGTOGCGCA GA---XTCAGC AO-TOC-SACA 300 C-03CGGACCC A--Α-XGAO--A TCAAGGCTCT GCOGGCAGAT CTCGGAGAGA TC AIG 355 Met 1 AGC Crc CCG C-W GAG CϊOG TCE TOG CCT TCA AGG CGA ACC CTC ACG TTG 403 Ser Leu Pro His Glu Pro Ser Ser Pro Ser Arg Arg Thr Leu Ihr Leu 5 10 15 ATC CTG GCC GCT GCT GCT GGT CIC GCA CIG GIG GCC GOC TOG ATC GTC 451 lie Leu Ala Ala Ala Ala Gly Leu Ala Leu Val Ala Ala Trp He Val 20 25 30 ATC GCC ACC AGG TOG TOG CCA 003 AGG ACT GCT CCC ACC ACA GAA GQC 499 He Ala Ihr Arg Ser Ser Pro Pro Ihr Ser Pro Pro Ihr Thr Glu Gly 35 40 45 QQC CAG GTC ACS ACC CCA GCC CCC AAC GAC CCC ACC GCC GTC ACC CCC 547 Gly Gin Val Thr T-hr Pro Ala Pro Asn Asp Pro Ihr Ala Val Thr Pro 50 55 60 65 GAG AGC CTC GCC TOG TOC G^ GAG Trc GAC GGC GCC CXE GGG TOG GCG 595 Glu Ser Leu Ala Trp Ser Asp Glu Fhe Asp Gly Ala Ala Gly Ser Ala 70 75 80 COS AAC CCC GAC GTS TOG AAC CAC GAG ACC GGC GCC GQC GGT TOG GGC 643 Pro Asn Pro Asp Val Trp Asn His Glu Thr Gly Ala Gly Gly Trp Gly 85 90 95 AAC GCC GAG CTC CAG AAC TAC ACG ACG TOG COG GTC AAC TOG GOG CTC 691 Asn Ala Glu Leu Gin Asn T r Thr Thr Ser Arg Val Asn Ser Ala Leu 100 105 110 GAC GGT CAG GGC AAC CIG GTC ATC ACC GOG CTC CAG GAG AGC GAC GGG 739 Asp Gly Gin Gly Asn Leu Val He Thr Ala Leu Gin Glu Ser Asp Gly 115 120 125 TOG T-AC AOG TOC GCA CGC TTG ACC ACG CAG GGC AAC GTC CAG COG CAG 787 Ser Tyr Thr Ser Ala Arg Leu Thr Thr Gin Gly Asn Val Gin Pro Gin 130 135 140 145 TTC GCT CGA ATA GAG GOG CGC ATC CAG ATC COG CCT GGC CAG GGC ATC 835 Ehe Gly Arg He Glu Ala Arg He Gin He Pro Arg Gly Gin Gly He 150 155 160 TOG TOC GOG TIC TOG AIG GTC GGA GGG - - CTC CJX GAC ACC CCE TOG 883 Trp Ser Ala R-e Trp Met Val Gly Ala Asn Leu Pro Asp Thr Pro Trp 165 170 175 CCT ACC TOC GCT GAG ATC GAC ATC ATG GAG AAC GIG GGC AAT GOG GCC 931 Pro Thr Ser Gly Glu He Asp He Met Glu Asn Val Gly Asn Ala Pro 180 185 190 CAC GAG GTC CAC GGC ACG GTC CAC GGG CCT GGG TAC TOC GGG GAC AAC 979 His Glu Val His Gly Thr Val His Gly Pro Gly Tyr Ser Gly Asp Asn 195 200 205 GGCATCATCGGCACCTACCAG CATCCG CAAGGGTOGTOGTTCG∞ 1027 Gly He Met Gly Thr Tyr Gin His Pro Gin Gly Trp Ser Phe Ala Asp 210 215 220 225 GACTrc C_ACA-XTrcGGC- :G--rTOGAα3Cσ3GCTGft3ATC 1075 Asp Phe His Thr B-e Gly He Asp T-rp Ihr Pro Gly Glu He Ihr Trp 230 235 240 CTC GIT GAC GGG CAG GAG TAT CAC CGC GTC AOG ACC GOG GAT GIC GCT 1123 Leu Val Asp Gly Gin Glu Tyr His Arg Val Thr Thr Ala Asp Val Gly 245 250 255 GCC AAC CAG TOG GTC TIC GAC CAG COG TTC TTC CTC ATC CTC AAC GTC 1171 Ala Asn Gin Trp Val Fhe Asp Gin Pro Phe Phe Leu He eu Asn Val 260 265 270 GCC ATC GGC GGC CAG TOG CCC GGC AAC CCC GAC GCA ACG ACC COG TTT 1219 Ala He Gly Gly Gin Trp Pro Gly Asn Pro Asp Ala Ihr Ihr Pro Phe 275 280 285 COG CAG CAG ATG AAG GTC GAC TAC GIG GGG GTC TAC GAC AAC GOG AOG 1267 Pro Gin Gin Met Lys Val Asp Tyr Val Arg Val Tyr Asp Asn Ala Thr 290 295 300 305 CAG TA--XX-AO0CT 0G0G0QGGCG OGGGOGGTIC GA OGGGAAC GGCT0GAGAC 1320 Gin GCTCGTCGCT GAAOGAOCAG OGICGAOCIG GGGCAA0CTG A--CGTIGCIG GIGCAGCIGG 1380 GICATOCTCT GACTOCGGGG TCTACGG0QG AIGO-CATOG TOGCIQOOGC COGGAGGGGC 1440 GGTAGGGGOG TCAGGTAO- 1459 (2) _NK-PMATiαN PCR SEQ ID NO: 2: (i) SEQUENCE CHA-Wπ-E ISTICS: (A) -LENS-H: 306 amino acids (B) TYPE: amino acid (D) TϋSOT-OGY: linear (ii) M- X-ULE TYPE: protein (xi) SEQUENCE D--SCRIPITCN: SEQ ID NO: 2: Met Ser Leu Pro His Glu Pro Ser Ser Pro Ser Arg Arg Ihr Leu Thr 1 5 10 15 Leu lie Leu Ala Ala Ala Ala Gly Leu Ala Leu Val Ala Ala Tcp He 20 25 30 Val He Ala. Thr Arg Ser Ser Pro Pro Thr Ser Pro Pro Thr Thr Glu 35 40 45 Gly Gly Gin Val Thr Thr Pro Ala Pro Asn Asp Pro Thr Ala Val Thr 50 55 60 Pro Glu Ser Leu Ala Trp Ser Asp Glu Fhe Asp Gly Ala Ala Gly Ser 65 70 75 80 Ala Pro Asn Pro Asp Val Trp Asn His Glu Thr Gly Ala Gly Gly Trp 85 90 95 Gly Asn Ala Glu Leu Gn Asn Tyr Thr Thr Ser Arg Val Asn Ser Ala 100 105 110 Leu Asp Gly Gin Gly Asn Leu Val He Thr Ala Leu Gin Glu Ser Asp 115 120 125 Gly Ser Tyr Thr Ser Ala Arg Leu Thr Thr Gin Gly Asn Val Gin Pro 130 135 140 Gin Fhe Gly Arg He Glu Ala Arg He Gin He Pro Arg Gly Gin Gly 145 150 155 160 He Trp Ser Ala Fhe Trp Met Val Gly Ala Asn Leu Pro Asp Thr Pro 165 170 175 Trp Pro Thr Ser Gly Glu He Asp He Met Glu Asn Veil Gly Asn Ala 180 185 190 Pro His Glu Val His Gly Thr Val His Gly Pro Gly Tyr Ser Gly Asp 195 200 205 Asn Gly He Met Gly Thr Tyr Gin His Pro Gin Gly Trp Ser Phe Ala 210 215 220 Asp Asp E*-e His Thr R-e Gly He Asp Trp Thr Pro Gly Glu He Thr 225 230 235 240 Trp Leu Val Asp Gly Gin Glu Tyr His Arg Val Thr Thr Ala Asp Val 245 250 255 Gly Ala Asn Gin Trp Val Phe Asp Gin Pro Fhe Fhe Leu He Leu Asn 260 265 270 Val Ala He Gly Gly Gin Trp Pro Gly Asn Fro Asp Ala Thr Thr Pro 275 280 285 Phe Pro Gin Gin Met lys Val Asp Tyr Val Arg Val Tyr Asp Asn Ala 290 295 300 Thr Gin 305 (2) INPCPMAHCN PCR SEQ ID NO: 3: (i) -SEQUENCE CHARACTERISTICS: (A) I-ENGIH: 181 base pairs (B) TYPE: 'nucleic acid (C) --T---t©----NESS: single (D) TOPOLOGY: linear (ii) M- EUL-E TYPE: ENA (gencmic) (iii) H--POT-_ETICAL: NO (iv) ANIT-SENSE: NO (vi) CRIGINAL SOURCE: (B) SI AIN: Oerskovia xanthineolytica IIG109 l rt q cra - fg gc αcagttcc agg g'=g^^ ac - q ^g^r'- ^g^g''''r<''7g'~g'~g^a gc , tg F V D G Q Q F H V T R E L G λ N A W 61 gtgttcgaccayccq C tcrt CC - catcctrr Λ acgtcgcggtcggcgggcagtggccgggc V F U Q P F P L I L N V A V G G Q W G .21 taccccgacgqr.a qacccaqctcco q ca q ca q atσaaσσtcσal. ac:α--:--gCTccr-:cr-as; Y P D G T T L P Q Q M K V D Y V R \ r γ loi a INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule \3bis) A. The indications made below relate to the microorganism referred to in the description on page 2 1 ' ine 33 - 36 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | Name of depositary institution DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELL¬ KULTUREN GmbH Address of depositary institution (including postal code and country) Mascheroder Weg lb, D-38124 Braunschweig, Federa l Re¬ public of Germany Date of deposit Accession Number 13 O cto b er 1995 DSM 10297 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 of Australia Statutory Rules 1991 No 71) . 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 natureoftheindication cg., "Accessio Number of Deposit") For International Bureau use only I I This sheet was received by the International Bureau on Authorized officer Form PCT RO/134 Jul 1992

Claims: PATENT CLAIMS 1. A DNA construct comprising a DNA sequence encoding an 5 enzyme exhibiting 3-1,3-glucanase activity, which DNA sequence a) comprises the DNA sequence shown in SEQ ID No. 1, or b) comprises an analogue of the DNA sequence shown in SEQ ID No. 1, which i) hybridizes with the same oligonucleotide probe as the DNA 10 sequence shown in SEQ ID No. 1, and ii) 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 iii) encodes a polypeptide which is immunologically reactive 15 with an antibody raised against a purified 0-1,3 glucanase encoded by the DNA sequence shown in SEQ ID No. 1 derived from Oerskovia Xanthineolytica LLG109. 2. The DNA construct according to claim 1, comprising a 20 DNA sequence encoding an enzyme exhibiting β-1,3-glucanase activity, which DNA sequence comprises the DNA sequence shown in SEQ ID No. 1. 3. The DNA construct according to claims 1 and 2, which 25 DNA sequence comprises the DNA sequence shown in SEQ ID No. 3. 4. The DNA construct according to any of claims 1 to 3, in which the DNA sequence is derived from a microorganism. 305. The DNA construct according to claim 4, in which the DNA sequence is derived from a filamentous fungi or a yeast. 6. The DNA construct according to claim 5, in which the DNA sequence is derived from a strain of the group comprising 35 Aspergillus , Trichoderma , Fusarium or Humicola . 1. The DNA construct according to claim 4, in which the DNA sequence is derived from a bacterium. 8. The DNA construct according to claim 7, in which the DNA sequence is derived from bacteria from the group comprising Bacillus , especially B . licheniformis , B . amyloliquefaciens , B . circulanε , or Cloεtridium, especially Cl . termocellum , or 5 Oerεkovia , Arthrobacter, Cytophaga or Rhodothermuε . 9. The DNA construct according to claim 8, in which the DNA sequence is derived from Oerskovia xanthineolytica , especially Oerεkovia xanthineolytica LLG109 (DSM no. 10297) . 10 10. A recombinant expression vector comprising a DNA construct according to any of claims 1 to 9. 11. The recombinant expression vector according to claim 1510, wherein the vector comprises the maltogenic α-amylase promoter from B . stearothermophilus . 12. The recombinant expression vector according to claims 10 and 11, wherein the vector comprises the maltogenic α- 20 amylase signal from B. εtearothermophiluε . 13. The recombinant expression vector according to any of the claims 10 to 12, comprising a sequence from the pPFFl plasmid. 25 14. A cell comprising a DNA construct according to any of claims 1 to 9 or a recombinant expression vector according to any of the claims 10 to 13. 3015. The cell according to claim 14, which cell is a microbial cell. 16. The cell according to claim 15, which cell is a bacterial cell. 35 17. The cell according to claim 16, in which the cell is a bacterial cell selected from the group comprising bacteria such as or Oerεkovia , Arthrobacter, Cytophaga , Rhodothermus , or strains of Bacilluε , such as strains of B. εubtiliε , B . licheniformiε, B . lentuε, B. Jbrevis, B . εtearothermophiluε , B . alkalophilus , B . amyloliquefaciens , B . coagulans , B . circulanε, B . lautuε , B . megaterium or B . thuringienεiε, or strains of 5 Streptomyces, such as S. lividans or S. murinus , or strains of Escherichia , such as E. coli . 18. The cell according to claim 16 and 17, which bacterial cell is Bacillus subtilis DN1885. 10 19. The cell according to claim 16 and 17, which bacterial cell is Bacillus subtilis ToC46. 20. The cell according to claims 14 and 15, in which the 15 cell is a bacteria cell different from the cell from which the DNA sequence originates. 21. The cell according to claims 14 and 15, in which the cell is a fungal cell. 20 22. The cell according to claim 21, in which the cell is a fungal cell selected from the group of a strain of Aspergillus , Trichoderma , Fusarium, or Humicola . 2523. The cell according to claims 14 and 15, in which the cell is a fungal cell different from the cell in which the DNA sequence originated. 24. A method of producing an enzyme exhibiting jS-1,3- 30 glucanase activity, comprising culturing a cell according to any of the claims 14 to 23, under conditions permitting the production of the enzyme, and recovering the enzyme from the culture. 3525. A novel enzyme exhibiting β-1 ,3-glucanase activity, which enzyme a) is encoded by a DNA construct according to any of claims 1 to 9, b) produced by the method according to claim 24. 26. The novel enzyme according to claim 25 which is is immunologically reactive with an antibody raised against a 5 purified 3-1,3-glucanase derived from Oerskovia xanthineolytica strain LLG109. 27. The novel enzyme according to claims 25 and 26, wherein the enzyme has an apparent molecular weight of about 26 KDa. 10 28. The novel enzyme according to any of claims 25 to 27, wherein the enzyme has a pi in the range of about 3.5 to 4. 29. An enzyme preparation useful for modification or 15 degradation of 0-glucan containing material, comprising a novel enzyme according to any of claims 25 to 28. 30. The enzyme preparation according to claim 29, comprising one or more other cell wall degrading enzymes, 20 selected from the group of enzymes having cellulytic, mannanolytic, chitinolytic or proteolytic activities. 31. The enzyme preparation according to claim 30, wherein said enzyme is selected from the group comprising endo- 25 glucanases, such as cellulases and 0-1,6-glucanases, and mannanases, endo- or exo- chitinases, proteases, and α- or β- mannosidases. 32. The enzyme preparation according to claim 30 and 31, 30 comprising an enzyme from the group comprising includes Novozy e® 234 and Cereflo® 200L, Cellulase, Cellulase CP and Cellulase CT, and/or Chitinase. 33. Use of an enzyme according to any of the claims 25 to 3528, in which the enzyme or enzyme preparation according to any of the claims 29 to 32 for the modification or degradation of a jS-glucan containing material. 34. The use according to claim 33, in which the enzyme or enzyme preparation is used in the preparation of protoplast. 35. The use of an enzyme according to claim 33, in which the enzyme or enzyme preparation is used in the preparation of pigments, colorants, flavorants, yeast extracts. 36. The use of an enzyme according to claim 33, in which the enzyme or enzyme preparation is used in the preparation of pharmaceuticals. 37. Use of an enzyme according to claim 33, in which the enzyme or enzyme preparation is used in pharmaceuticals.

Inventors: ASENJO JUAN A, DIERS IVAN, FERRER PAU, HALKIER TORBEN, HEDEGAARD LISBETH, SAVVA DEMITRIS, ASENJO, JUAN, A., DIERS, IVAN, FERRER, PAU, HALKIER, TORBEN, HEDEGAARD, LISBETH, SAVVA, DEMITRIS
Applicants: ASENJO JUAN A, DIERS IVAN, FERRER PAU, HALKIER TORBEN, HEDEGAARD LISBETH, NOVONORDISK AS, SAVVA DEMITRIS, ASENJO, JUAN, A., DIERS, IVAN, FERRER, PAU, HALKIER, TORBEN, HEDEGAARD, LISBETH, NOVO NORDISK A/S, SAVVA, DEMITRIS
Application year: 1995
Main classification: C12N9
IPC codes: A61K38, C12N1, C12N9, C12N15, C12R1