JPH10507078A - Novel enzyme having β-1,3-glucanase activity - Google Patents

Abstract

  (57) [Summary] The present invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting β-1,3-glucanase activity, said DNA sequence comprising: a) the DNA sequence shown in SEQ ID NO: 1; 1) comprising an analog of the DNA sequence shown in SEQ ID NO: 1, said analog hybridizing with (i) the same oligonucleotide probe as the DNA sequence shown in SEQ ID NO: 1; A DNA sequence that encodes a polypeptide homologous to the polypeptide encoded by the DNA sequence comprising the indicated DNA sequence, or (iii) the DNA sequence set forth in SEQ ID NO: 1 derived from Oerskovia Xanthineolytica LLG109 Encodes a polypeptide that is immunologically reactive with antibodies raised against the purified β-1,3-glucanase encoded by Further, the present invention provides an expression vector comprising the DNA construct, a cell harboring the DNA construct or the expression vector, a method for producing a novel enzyme, a novel enzyme and a preparation containing the novel enzyme, It relates to the use of enzymes for degrading or altering glucan-containing substances.

Description

DETAILED DESCRIPTION OF THE INVENTION Novel enzyme having β-1,3-glucanase activity Field of the invention   The present invention relates to a novel enzyme exhibiting β-1,3-glucanase activity. further Specifically, a DNA construct encoding a novel enzyme, and a DNA construct comprising the DNA construct A cell comprising the current vector, the DNA construct or the recombinant expression vector, A method for producing the novel enzyme, an enzyme preparation comprising the novel enzyme, and Use of enzymes to decompose or alter substances containing .beta.-glucan . Background of the Invention   A large and diverse group of non-motile, heterotrophic, eukaryotes are collectively called fungi ing. Most fungi are saprophytes, that is, they feed from dead biological material. To win. Heterotrophic, that is, using biological material as a carbon source Many fungi produce metabolites of industrial importance. Some are food sources Other species are more useful, but other species are May cause rottenness.   The size of a fungal cell can range from a microscopic unicellular organism to a macroscopic one, for example, a mushroom. Range. True fungi are generally terrestrial, and zygomycetes (Zygomycet es), for example, Rhizopus, Basidiomycetes, for example, Puccinia graminis, Ascomycetes, for example , Neurospora and Saccharomyces and And Deuteromycetes, such as Penicillium and It is Aspergillus (Aspergillus).   Fungal cells   Fungal microorganisms include multicellular as well as unicellular organisms, and generally include yeast and It is thought to consist of mold. Unicellular fungi are mainly yeast, The term is mainly used for fungi that are mycelium.   Fungal cells have a very complex structure and are built from the cytoplasm, Andria, microbodies, etc., are enveloped by the cytoplasmic membrane. Conversion Chemically and structurally, the cytoplasmic membrane is a phospholipid with different proteins inserted into it. It consists of two layers of quality.   The cytoplasmic membrane is surrounded by rigid cell walls.   Fungal cell wall structure   The cell wall of most fungal microorganisms has a glucan network that provides the strength of the cell wall. contains. In addition, the major fungal cell wall components are mannoproteins and kites. It is.   Glucans and chitin are found in a number of major fungal-like organisms, such as Oomycetes (Oomycet es) is more resistant to microbial degradation than cellulose, a major component of the cell wall. It is even more resistant.   Glucan is mainly β-1,3 linked, and some It has a branch (Manncrs et al., Biotechnol. Bioeng., 38, p. 977, 1973), and It is known that it can be degraded by certain β-1,3-glucanase systems.   β-1,3-glucanase also has an endo-β- called laminalinase. Includes the group of 1,3-glucanases (EC 3.2.1.39 and EC. 3.2.1.6, Enzyme Nomenclature, Academic Press, Inc. 1992).   Pegg et al., Pysiol. Plant Pathol., 21, p. 389-409, 1982. Purified endo-β-1,3-glucanase and fungal exo-β-1, In combination with 3-glucanase, the fungus Perticillium arboatrum ( Verticillium alboatrum) has been shown to be able to hydrolyze the isolated cell wall .   Further, Keen et al., Plant Physiol., 71, p. 460-465, purified from soy Β-1,3-glucanase can degrade the isolated cell wall of fungi. Was done.   Large-scale degradation of fungal cell walls   The unit operation of cell disruption involves separation of intracellular products and downstream of valuable intracellular products. Appears as an essential first step for processing.   Large-scale cell disruption generally involves the use of strong chemical and / or mechanical means. Or rather by vigorous processing. This is very complicated of pollutants Leave the target protein with a good mixture.   In this regard, another approach to conventional microbial cell disruption techniques is the industrial Implementation has been adapted to increase adequacy (Asenjo et al., Bio / Technol., 11, p. . 214, 1993; De la Fuente et al., Appl. Microbiol. Biotechnol., 38, p. 7 63, 1993).   Bioprocessing by simplifying downstream processing of intracellular products As a means of increasing productivity, economics and product quality, selective cell permeabilization ( Selective Cell Permeabilization (SCP) and Selective Protein Recovery (Select) ive Protein Recovery (SPR) is highly Proven attractive (Asenjo et al., 1993, supra: Shen et al., Gene, 84, p. 1989).   SCP and SPR increase the porosity of the fungal cell wall (very limited cell lysis) Cell wall degradable β-glucose to promote the release of intracellular proteins. It involves using a pure preparation of canase. In this way, the SCP Gives a major separation of the primary product from some of its major contaminants. This approach Major limitations are currently obtained in bacteria used for the production of yeast lytic enzymes A relatively low level of expression of the enzyme (e.g., Elscobia Oerskovia Xanthineolytica, Andrews and Asenjo, Biotech . Bioeng. 30, p. 628, 1987). Today, Elscovia Xanthin Orichka (Oers kovia Xanthineolytica) is sometimes called Cellulomonas Cellance lulans). However, in the following the name Elscobia Xanthi Use Nitrichika.   Many useful commercial enzyme compositions are available for enzyme lysis of fungal cells . Such products usually comprise a large number of enzymatic activities, and such enzymatic activities Are, for example, β-1,3-glucanases and β-1,6-glucanases, Includes proteases, chitinases, mannases and other enzymes that can degrade cell wall components Include.   Elscovia xanthine orichika LLG109 dissolution system   The lytic enzyme system of Elscovia xanthine orlitica LLG109 was partially isolated and Purified and characterized for some components of glucanase and protease (Ventom and Asenjo, Enzyme Microb. Technol., 13, p. 71, 1991).   The single molecular species of soluble β-1,3-glucanase is Elscobia xanthino. Characterized from Ritica LLG109, most Elscobia have a large number of β-1,3 -Seems to have a glucanase system (Doi and Doi, J. Bacteriol., 16 8, p. 1272, 1986).   All observed molecular forms of these enzymes are associated with β-1,3-glucan. Hydrolysis activity (β-1,3-glucanase activity), but slightly Alone readily solubilizes yeast glucan and induces lysis of viable yeast cells Can be   In contrast, other types of endo-β-1,3-glucanase have glucans partially Solubilization only and will cause only limited cell lysis (Doi and Doi, supra, 1986). This large number of enzyme species produced by Elscovia It may be due to proteolytic processing.   The number of yeast lytic enzymes cloned so far is very limited, The genetic relationship between these enzymes is still unclear. As a result, the gene structure and Knowledge of the relationship to protein and protein function is still limited (Shen et al., JB iol. Chem. 266, p. 1058, 1991; Shimol and Tademura, J.M. Biochm., 110, 60 8, 1991: Watanabe et al. Bacteriol., 174 (1), p. 186, 1992: Yamamoto et al. Biosci. Biotechnol. Biochem., 57, p. 1518-1525, 1993).   Characterization of yeast lytic enzymes from Elscovia xanthine orythica   Purified soluble β-1,3-glucanase was estimated by SDS-PAGE. As a result, it showed a molecular weight of about 31 KDa and a pI of 5.0. Also, the amino acid composition is determined Was. The yield was optimized by the continuous culture method, but the yield was still low.   The specific activity of this enzyme was 11.1 U / mg. Β-1,3- against yeast glucan Km for glucanase activity is laminarin (soluble β-1,3-glucan) 2.5 mg / ml for 0.95 mg / ml. About β-1,3-glucanase The pH optimum is 8.0 for yeast glucan and 6.0 for laminarin substrate. Met. Dissolution Degradable β-1,3-glucanase is a viable yeast (Saccharomyces cerevisiae) (Saccharomyces cerevisiae)) causes only limited lytic activity on cells (Ventom and Asenjo, supra, 1991), which is a soluble protease component. Stimulated synergistically.   In addition, continuous fermentation broth of purified Elscovia xanthin orythica Other β-1,3-glucanase components were detected in it, but it was purified to homogeneity. And were subsequently characterized.   Patent literature   A number of β-1,3-glucanase genes and their uses have been patented. I have.   One example is DD226012 (Akad. Wissenshaft DDR), which is based on Bacillus (B. acillus), a process for producing β-1,3-glucanase.   Further, JP61040792A (DOI K) discloses a method for removing yeast cell walls. A cell wall citase β-1,3-glucanase recombinant plasmid has been described. . The gene was derived from Arthrobacter and was (richia) formed in bacteria.   EP 440,304 discloses intracellular chitinase or intracellular or extracellular β- Disease transformed with at least one gene encoding 1,3-glucanase The present invention relates to plants having improved resistance to protozoal fungi. In addition, matching recombination Polynucleotides are disclosed.   WO 87/01388 describes cell lysis that can be produced by Elscobia. A method for producing a protease, for example, β-1,3-glucanase, is described.   WO92 / 03557 discloses that it is derived from Elscovia xanthine orythica, Recombinant DN comprising a 2.7 Kb DNA sequence encoding β-1,3-glucanase A expression vectors are disclosed. The glucanase is expressed in E. coli. Shows glucanase activity but no protease activity.   E. coli has a number of disadvantages for large-scale industrial enzyme production. First of all Lucanase is expressed intracellularly. After all, to gain access to the enzyme, It is necessary to open the cell. This makes recovery of the enzyme difficult and expensive. You.   Recombination encoding a novel protein having β-1,3-glucanase activity The DNA sequence is known from WO 92/16632. This gene is derived from soy Is done.   Description of the prior art   Most currently available enzyme preparations used to lyse fungal cells are protein Activity, which can be dissolved with a very complex mixture of contaminants. Leave the target protein.   Therefore, it contains substantially no protease activity and has a gentle It is desirable to provide a β-1,3-glucanase that is able to open. this Facilitates recovery and purification of the target protein.   Furthermore, in heterologous host cells capable of increasing the production yield, the target protein It may be advantageous to express a gene encoding Summary of the Invention   Surprisingly, by providing a novel β-1,3-glucanase Successfully solved some of the aforementioned problems.   First, a novel 26 kDa that exhibits β-1,3-glucanase activity A novel DNA sequence encoding this enzyme was isolated and characterized. According to the invention Then, a novel single-component β-1,3-glucanase can be produced.   Thus, in a first aspect, the present invention provides for β-1,3-glucanase activity. A DNA construct comprising a DNA sequence encoding a novel enzyme. The columns are   a) comprising the DNA sequence shown in SEQ ID NO: 1, or   b) comprises an analog of the DNA sequence shown in SEQ ID NO: 1, said analog comprising:   i) oligonucleotide probe and hive identical to the DNA sequence shown in SEQ ID NO: 1 Redistribution, and   ii) a poly-encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID NO: 1. Encodes a polypeptide that is homologous to the peptide, or   iii) SEQ ID NO: 1 derived from Elscovia xanthine orythica LLG109 For the purified β-1,3-glucanase encoded by the DNA sequence shown. Encodes a polypeptide that is immunologically reactive with the resulting antibody.   Elscovia Xanthin Oryka LLG109 is in compliance with the Budapest Treaty E Samrung von Microorganizmen und Zerkulzlen (De utshe Sammlung von Mikroorganismen und Zcllkulturen) GmbH. (DSM) Was.   In the context of the present invention, the expression “analog” of the DNA sequence shown in SEQ ID NO: 1 Exhibiting β-1,3-glucanase activity and having the above properties i) to iii) Is intended to indicate any DNA sequence that encodes Typically, similar DNA sequences The columns are -Based on any of the DNA sequences shown in SEQ ID NO: 1 Using the techniques described herein, have β-1,3-glucanase activity. Other enzymes that are known to produce, or are thought to produce, enzymes that Or is isolated from a related (eg, identical) organism, or -Based on any of the DNA sequences shown in SEQ ID NO: 1 Produces no other amino acid sequence of the encoded β-1,3-glucanase, Derivation of nucleotide substitutions corresponding to the codon usage of the host organism intended for elemental production Or different amino acid sequences, and therefore, probably Different proteins producing β-1,3-glucanase variants having different properties from Constructed by the introduction of nucleotide substitutions resulting in a conformational structure. Other possible modifications Examples are the insertion of one or more nucleotides into a sequence, any of the sequences The addition of one or more nucleotides at the termini, or Or deletion of one or more nucleotides in the sequence. For example, A similar DNA sequence indicates that the encoded protein has a corresponding β-1,3-gluca. It can be a subsequence of the DNA sequence shown in SEQ ID NO: 1 that exhibits a kinase activity.   The hybridization referred to in the above i) is carried out by the following materials and methods. Under certain specified conditions, as described in detail in the section below, a novel 26 kDa β- For a probe identical to the DNA sequence encoding the 1,3-glucanase enzyme, It is intended to indicate that similar DNA sequences hybridize.   Usually, similar DNA sequences are highly homologous to the DNA sequence; At least 60% relative to the above sequence encoding β-1,3-glucanase Homologous, for example, at least 70% less than the sequence shown in SEQ ID NO: 1 At least 75%, at least 80%, at least 85%, at least 90% or even at least 95% homologous.   The degree of homology mentioned in ii) above indicates the derivation of the second sequence from the first sequence. It is determined as the degree of identity between two sequences. Homology is known in the art. Computer programs, such as the GCG program package Appropriate decisions can be made by the GAP provided (Needleman, SB and Wu nsch, C.I. D., (1970), Journal of Molecular Biology, 48, 443-453). DNA distribution The following settings for row comparison: GAP occurrence penalty of 5.0 and GAP of 0.3 Using a GAP with an extension penalty, the coding region of the DNA sequence An enzyme encoded by a DNA construct comprising DNA SEQ ID NO: 1; Have a degree of identity of at least 60%, for example, at least 75% or 90% .   The term "derived from" with respect to the properties of iii) above refers to Elscobia xanthan Only showing β-1,3-glucanase produced by L. DNA sequence isolated from Elscovia xanthine Orytica LLG109 Sequence and is transformed with a vector comprising said DNA sequence. Β-1,3-glucanase produced in a living host organism . Immunological reactivity was determined by the methods described in the Materials and Methods section below. Can be specified.   In another aspect, the invention relates to the construction of an expression vector harboring the DNA construct of the invention. Cells comprising the DNA construct or expression vector, and a β-1,3-glu A method for producing a novel enzyme exhibiting canase activity, the method comprising: Culturing the cells under conditions that permit and recovering the enzyme from the culture. Comprising.   In still another aspect, a novel compound exhibiting β-1,3-glucanase activity is disclosed. The enzyme,   a) encoded by the DNA construct of the present invention;   b) produced by the method of the invention and / or   c) as shown in SEQ ID NO: 1 derived from Elscobia xanthine orlitica LLG109 To purified β-1,3-glucanase encoded by the DNA sequence Immunologically reactive with the same antibody.   The invention also relates to the degradation of substances containing β-glucan, in particular microbial cell wall substances. Alternatively, for enzyme preparations useful for modification, the composition may comprise β-1 as described above. , 3-glucanase activity.   Finally, for the preparation of fungal protoplasts or fungal extracts, especially yeast extracts The use of novel enzymes or enzyme preparations is also contemplated in the present invention.   A final object of the present invention is to transfer the fungal cells in question to a novel β-1,3-glucanase. Or recovery of biological components from fungal cells by exposure to the enzyme preparation Is to provide a way to BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows a map of the plasmid pPFF1.   FIG. 2 shows a map of the plasmid pPF8A.   FIG. 3 shows the predicted and partially determined amino acid sequences. 1 shows a 1.5 kb BamHI-KpnI according to the invention in comparison.   FIG. 4 shows a map of the plasmid pPF15BK.   FIG. 5 shows a map of the plasmid pPF11BgK. Detailed description of the invention   The present inventors have now surprisingly found that they substantially contain protease activity. And succeeded in providing a novel β-1,3-glucanase. In the desired cell Use of novel β-1,3-glucanases to gain access to components Facilitates the recovery process. Novel enzymes are delicate in a gentle way The vesicle is opened and the method involves forming large, multiple pores in the cell wall. Sa Furthermore, this method reduces the amount of pollutants.   Furthermore, the present inventors have developed novel β-1,3-glucose in suitable heterologous host cells. Expression of the gene encoding canase was achieved. For details, In B. subtilis, suitable for industrial production, a novel β-1 , 3-glucanase yield is increased compared to the parent cell from which the gene is derived.   The DNA sequence of the present invention, which encodes an enzyme exhibiting β-1,3-glucanase activity, comprises: It can be isolated by a general method comprising the following steps: -In a suitable vector, from Elscovia xanthine Orythica LLG109 Clone the DNA construct, Transforming said vector with a suitable host cell, -Culturing the host cells under suitable conditions that express any enzyme of interest; The β-1,3-glucanase activity of the enzyme produced by such a clone Screening for positive clones by determining -Select a clone, and -Isolating the DNA-encoding enzyme from such clones.   The general method is described in WO 93/11249, which is hereby incorporated by reference. Is further disclosed. This method is described below This will be described in more detail.   The DNA sequence encoding the enzyme is, for example, that of Elscovia xanthin orythica. LLG109 strain (Lechevalier, Int. J. Sys. Bacteriol., 22 (4), p. 260, 1972), and select for clones that express appropriate enzyme activity (Ie, a suitable substrate such as laminarin or AZCL-co). Β determined by the ability of the enzyme to hydrolyze the β-1,3-glucan bond of rudran -1,3-glucanase activity, see Materials and Methods section below).   Elscovia Xanthin Oryka's LLG109 strain has been In accordance with the provisions of the Budapest Treaty on the international recognition of the deposit of microorganisms, Zamrun von Microorganizmen und Zerkulzlen (Deutsh e Sammlung von Mikroorganismen und Zellkulturen) GmbH. (DSM) . Deposit date: October 13, 1995. Depositary reference: NN049107 ((Oerskovia Xanthineoly tica LLG109). DSM display: Oerskovia Xanthineolytica (or Cellulomonas Cell ulans) DSM No. 10297.   Deutsche Zamrun von Micro-Organizmen und Selkultz Ren GmbH is the authority for international deposits under the Budapest Treaty, And the provision of the permanence of the deposit in accordance with the regulations, in particular see Rule 9. Access to the deposit Seth was the Director of the United States Patent and Trademark Office while the patent application was pending. 37C. F. R. Par. 1. 14 and 35 U.S. S. C. Par. Grant rights under 122 It is possible for what is determined to be obtained. Also, the aforementioned deposits are governed by Rule 28EP Satisfies the requirements of European patent applications for microorganisms according to C.   The foregoing deposit represents a substantially pure culture of the isolated bacterium. Foreign patent law in the country where the subject application is filed or its equivalent is filed. Deposits are available when more required. However, the entry of the deposited strain The possibility of the revocation of a patent granted by a government activity is subject to the invention of the subject matter. It should be understood that there is no implementation contract for implementing   Then use standard techniques, for example, as described in the Materials and Methods section. Thus, the appropriate DNA sequence can be isolated from the clone.   According to the present invention, the DNA construct comprises a DNA sequence, said DNA sequence comprising:   a) comprising the DNA sequence shown in SEQ ID NO: 1, or   b) comprises an analog of the DNA sequence shown in SEQ ID NO: 1, said analog comprising:   i) oligonucleotide probe and hive identical to the DNA sequence shown in SEQ ID NO: 1 Redistribution, and   ii) a poly-encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID NO: 1. Encodes a polypeptide that is homologous to the peptide, or   iii) SEQ ID NO: 1 derived from Elscovia xanthine orythica LLG109 For the purified β-1,3-glucanase encoded by the DNA sequence shown Encodes a polypeptide that is immunologically reactive with the resulting antibody.   In an embodiment of the present invention, the DNA construct has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. Comprising.   Polypeptide encoded by the DNA sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3 The tide is shown in SEQ ID NO: 2 and SEQ ID NO: 3.   A preferred method of amplifying a specific DNA sequence is to use synthetic degenerate oligonucleotides. This is a method using a polymerase chain reaction (PCR) using a probe.   For example, PCR is described in U.S. Pat. K. Saiki et al. (1988) , Science, 239, 487-491.   Is the DNA sequence encoding the homologous enzyme, that is, a similar DNA sequence Is expected to be obtained. For example, DNA sequences may be It can be derived from fungi or bacteria.   Such DNA sequences can be found in strains of the species Aspergillus, especially Aspergillus acres. Atus (A. aculeatus), strain of Aspergillus niger, Strains of the Trichoderma species, especially T. reesie, Trichoderma viride (T. viride), Trichoderma longibrachiatum (T. lo ngibrachiatum) or Trichoderma koningii (T. koningii), Trichoderma Strains of T. harzianum or Fusarium species, Strains of Fusarium oxysporum (F. oxysporum) or Humicola (Humico) la) can be derived from fungi comprising strains of the species.   In addition, the DNA sequence encoding the homologous enzyme can be modified using other strains of Elscovia or Arthrobacter species, Cytophaga strain, Rhodotherm Rhodothermus strains, especially Rhodmarinus strains Or Clostrium, in particular, Clostorium sermoserum (Cl. The rmocellum), strains of the Bacillus species, especially Bacillus riheniphor Miss (B. licheniformis), Bacillus amyloliquefaciens ( B. amyloliquefaciens) or Bacillus circulans It is expected that it can be derived from the strain.   The DNA encoding the β-1,3-glucanase of the present invention is a well-known DNA. In accordance with the techniques described herein, DNA from any of the aforementioned organisms is described herein. Using synthetic oligonucleotide probes prepared based on the DNA sequence Can be isolated. For example, suitable oligonucleotide probes are It can be prepared based on the partial nucleotide sequence shown in column number 1.   Subsequently, the DNA sequence can be inserted into a recombinant expression vector. This It can be any vector that can be conveniently applied to recombinant DNA techniques. And the choice of vector often depends on the host cell into which it is to be introduced. There will be. Thus, vectors are autonomously replicating vectors, i.e., chromosomes. A vector that exists as a foreign entity and whose replication is independent of chromosomal replication For example, it can be a plasmid. In addition, the vector is contained in the host cell. Upon introduction, it is integrated into the genome of the host cell, and one of the integrated Or can be replicated together with two or more chromosomes.   In the vector, the DNA sequence encoding β-1,3-glucanase is Should be operably connected to the appropriate promoter and terminator sequences . A promoter is any DNA sequence that exhibits transcription activity in a selected host cell. And encodes a protein homologous or heterologous to the host cell. Can be derived from the gene.   Bacillus stearothermophilus and / or Or maltogenic from the signal of Bacillus stearothermophilus ( maltogenic) of β-1,3-amylase It is preferred to use the vector under the control of a promoter for the purpose.   In certain embodiments, the expression vector comprises the plasmid pPFF1 shown in FIG. Become.   The DNA sequence encoding β-1,3-glucanase, the promoter and And terminator, and insert them into an appropriate vector. The techniques used for this are well known to those skilled in the art (eg, Sambrook et al., Molecula r Cloning: See A Laboratory Manual, Cold Spring Harbor, NY, 1989. When).   A host cell transformed with a DNA sequence encoding the enzyme of the present invention can be a eukaryotic cell. Or a prokaryotic cell.   Suitable prokaryotic host cells are in particular bacterial cells.   Such a bacterium host capable of producing the novel enzyme of the present invention when cultured. Examples of primary cells include gram-positive bacteria, for example, strains of Bacillus, for example, Ciris, Bacillus ligeniformis, Bacillus lentus, Bashi Las Brevis, Bacillus stearothermophilus, Bacillus B. alkalophilus, Bacillus amyloliquefacier Bacillus coagulans, Bacillus circulans , B. lautus, B. megaterium, etc. Or a strain of B. thuringiensis or streptococcus Streptomyces, such as Streptomyces lividans ns) or a strain of Streptomyces murinus, or gram shade Sex bacteria, for example, E. coli. Bacterial transformation is protoplast transformation Or use competent cells in a manner known per se Can be implemented by . Bacterial transformation may be by protoplast transformation or by itself. Can be performed by using competent cells in a (See Sambrook et al., Supra, 1989).   When expressing an enzyme in a bacterium, for example, E. coli, the polypeptide is located in the cytoplasm. , Typically retained as insoluble granules (known as inclusion bodies) or Or it can be directed to the periplasmic space by bacterial secretory sequences. In the case of the former By lysing the cells, collecting and denaturing the granules, and then diluting the denaturant. To refold the polypeptide. In the latter case, the cells are Disintegrate by sonication or osmotic shock to dissolve the contents of the periplasmic space. Release and recover the polypeptide to recover it from the periplasmic space. Can be collected.   In certain embodiments of the invention, the bacterial host cell is a strain of Bacillus subtilis, particularly Bacillus subtilis DN1885 strain or protease-deficient Bacillus subtilis ToC4 There are six shares.   Suitable eukaryotic cells are in particular fungal cells, for example yeast or filamentous fungal cells.   Examples of suitable yeast cells are Saccharomyces species, especially Saccharomyces cerevisiae. , Saccharomyces kluyveri, Saccharomyces Ubalam (Saccharomyces uvarum) or Schizosaccharomyces (Schizosacchar) omyces) species, such as Schizosaccharomyces pombe Cells.   Yeast cells are transformed with heterologous DNA, and heterologous polypeptides are produced therefrom. Methods are described, for example, in the following documents: US Pat. No. 4,599,311; National Patent No. 4,931,373, U.S. Patent No. 4,8 Nos. 70,008, U.S. Pat. No. 5,037,743, and U.S. Pat. All of which are incorporated herein by reference). Normal drug resistance The absence of specific nutrients, such as leucine Transformed cells by a phenotype determined by their ability to grow in select. A preferred vector for use in yeast is U.S. Pat. No. 373, the POT1 vector. The polypeptide of the present invention Before the DNA sequence to be loaded, for example, as described above, the signal sequence and Optionally, a leader sequence can be present. Another example of a suitable yeast cell is Kluyveromyces species, for example, Kluyveromyces lactis (Kluyv eromyces lactis), or Hansenula species, such as Hansenula po Limorpha (Hansenula polymorpha) or Pichia species, for example, Chia pastoris (Pichia pastoris), Yarrowia species, for example, A strain of Yarrowia lipolytica (Gleeson et al., J. Gen. . Microbiol. 132,1986, pp. 3459-3465; see U.S. Pat. No. 4,882,279. See).   Examples of other fungal cells include filamentous fungi, such as Aspergillus sp., Neurospora sp. , Fusarium species, or Trichoderma species, especially Aspergillus oryzae (A. oryz) ae), Aspergillus nidulans or Aspergillus nidulans It is a cell of Russ Niger strain. Using Aspergillus for protein expression For example, European Patent (EP) 272,277, European Patent (EP) 238,023 and And EP 184,438. Fusarium Oxys Polam is described, for example, in Malardier et al., Gene 78, p. 147-156, 1989 It can be implemented as follows.   When a filamentous fungus is used as a host cell, it is conveniently located within the host chromosome. By obtaining the recombinant host cell by incorporating the DNA construct of the present invention into the DNA construct, It can be transformed with a building. This integration ensures that the DNA sequence is stable in the cell It is generally considered advantageous because it seems to be maintained. DNA constructs Integration into the host chromosome is performed according to conventional methods, for example, by homologous or heterologous recombination. Can be implemented.   In yet another aspect, the invention relates to a method for producing an enzyme, wherein the method comprises the steps of: The appropriate host cell transformed with the DNA sequence encoding the enzyme to allow production of the enzyme. The cells are cultured under operable conditions, 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. For example, a minimum medium or a complex medium containing appropriate auxiliary components. Appropriate Media is available from commercial suppliers or published formulations (eg, in the catalog of the American Type Culture Collection). Can be.   The expressed β-1,3-glucanase produced by the cells is then added to the medium. Can be recovered by a conventional method. Conventional techniques include centrifugation or filtration, salting Precipitation of protein components of the supernatant or filtrate with, for example, ammonium sulfate, seeds Various chromatographic techniques, for example depending on the type of polypeptide in question And ion exchange chromatography, gel filtration chromatography, affinity Host cells are separated from the culture medium by purification, such as by chromatography. It is included.   In yet another aspect, the present invention provides a method for modifying or separating a substance containing β-glucan. With respect to a novel enzyme preparation useful for the solution, said preparation comprises a β-1,3- Concentrate enzymes showing glucanase activity Have.   An enzyme preparation enriched in the novel β-1,3-glucanase enzyme of the present invention is described in Examples For example, enzyme preparations comprising a large number of enzymatic activities, in particular the modification of microbial cell walls or It can be an enzyme preparation comprising the different enzymatic activities required for degradation.   Examples of such enzyme preparations include lytic enzyme systems, especially those derived from microorganisms (fungi or bacteria). Conventional lytic enzyme synthesis, eg, Trichoderma, eg, Trichoderma harzianu Strains of Trichoderma viride or Trichoderma ricies, Elskobia For example, Elscobia xanthine orythica (sometimes Cellulomonas cellulans) (Called Cellulomonas Cellulans), a species of Arthrobacter, for example , A strain of Arthrobacter luteus, Rhizoctonia (Rhiz octonia) or Cytophaga, Staphyl. ococcus) strain or a lytic enzyme system derived from a Streptomyces strain Is included.   Commercially available enzyme preparations that can be conveniently facilitated with the enzymes of the invention / S, available from Denmark), Cellulase (available from Merk) Cellulase CP and Cellulase CT (both are available from Sturge), And / or Chitinase (available from Sigma), Zymolase (Kirin Brew eries).   In the context of the present invention, the term "concentrate" is advantageously used for a book prepared by the method described above. Increased β-1,3-glucanase activity of the enzyme preparation due to the addition of the enzyme of the invention Indicate, for example, that it is increasing at an enrichment factor of at least 1.1. Intended.   In addition, enzymes showing β-1,3-glucanase activity are concentrated. Enzyme preparations are preparations comprising the enzyme of the invention as the main enzyme component, for example, It can be a one-component enzyme composition.   Enzyme preparations can be prepared according to methods known in the art, And can be in the form of a liquid or dried preparation. For example, enzyme preparation The object can be in the form of granules or microgranules. Yeast to be included in the preparation The element can be stabilized by methods known in the art.   The enzyme preparation of the present invention comprises, in addition to the β-1,3-glucanase of the present invention, Can be used to degrade two or more other cell wall degrading enzymes such as cellulolytic, mannose, Enzymes having a proteolytic or proteolytic activity, such as endo- and So-glucanases, mannases, endo- and exo-chitinases, protea Or α- or β-mannosidase. Additional one Or two or more enzymes are of the genus Aspergillus, preferably Aspergillus niger , Aspergillus acreatus, Aspergillus awamori mori) or Aspergillus oryzae, or Trichode Luma, or Elscobia, Cytofaga, or Arthrobacter Microorganisms belonging to, or any of the aforementioned microorganisms, and commercially available enzyme preparations It can be produced in combination with an object.   Examples of preferred uses of the enzyme preparation according to the invention are given below. Departure The dosages of the bright enzyme compositions and conditions for using the compositions are known in the art. Can be determined based on the method being used.   The enzyme preparation according to the invention is preferably a substance containing β-glucan, such as e.g. For example, it is used as a decomposing or modifying agent for the cell wall of microorganisms. In particular, the yeast of the present invention Elemental preparations destroy the cell walls of microorganisms or Lysing, thereby allowing the recovery of the desired product produced by the microorganism Can be used for   The specific composition of the enzyme preparation to be used depends on the composition of the cell wall to be disrupted or lysed. It will be appreciated that it should be adapted. For example, there are two yeast cell walls The outer layers and the inner glue of the protein-mannan complex. And a can layer. To destroy yeast cell wall efficiently In addition, the enzyme preparation has at least protease activity, mannase and β-glucana. Desirably, it comprises a protease activity.   The extract recovered after disruption of the microbial cell wall usually contains a number of different components, e.g. For example, they comprise pigments, vitamins, colorants and flavors. Obtained from the destruction of yeast The extracted extract, i.e., yeast extract, can be used as such, Or its components can be recovered and further processed as necessary it can.   Examples of such products include vitamins, colorants (eg, carotenoids, Q-10 and And astaxanthin), enzymes, proteins and flavoring ingredients or flavor enhancers Agents (e.g., MSG, 5'-GMP and 5'-IPM). What products should be collected A unique product of the subject microorganism or the microorganism produces, for example, a recombinant product As such, it can be a constructed product.   In addition, the enzyme preparations of the present invention may be prepared from yeast (eg, Saccharomyces) Zosaccharomyces sp.) Or fungi (eg, Aspergillus sp. Um species). Such a creature The preparation and regeneration of these protoplasts involves fusion, transformation and cloning. Important in research. Protoplast production is known in the art. It can be carried out according to the following method.   The invention can also be used to improve fungal transformation.   Furthermore, the enzymes or enzyme preparations according to the invention can be used in preparations, in particular in In the preparation of products trapped inside cells in space and / or cell wall Can be used.   In addition, the enzyme preparations of the present invention may comprise β-glucans such as Kurdulane and Lami Can be used in changing Narin.   The following examples further illustrate the invention. These examples are for anyone Nor is the law intended to limit the scope of the invention.   Materials and methods   Donor organism:   Elscovia xanthine orlitica LLG109 (M. Lechevalier, Int. J. Sys. B acteriol. 22 (4), p. 260, 1972).   Elscobia xanthine orythica 73/14 (Shen et al., J. Biol. Chem. 266, p. 1058, 1991).   Deposited organism:   Elscovia Xanthin Oryka LLG109 strain is Deutsche Zamlung For Micro organizmen und Zerkulzulen (Deutshe Sammlung von M ikroorganismen und Zellkulturen) GmbH. (DSM).   Deposit date: October 13, 1995.   Depositary reference: NN049107 ((Oerskovia Xanthineolytica LLG109).   DSM display: Oerskovia Xanthineolytica (or Cellulomonas Cellulans) DSM N o. 10297.   Host organism:   Bacillus subtilis DN1885 (Diderichen et al., Journal of Bacteri ology, vol. 172, p. 4315-4321, 1990).   Bacillus subtilis protease deficient strain ToC46 (Diderichsen et al., Supra, 1990) .   E. coli JM109 (Yanish-Perron et al., Gene 33, p.103-199, 1985).   PF8A: E. coli JM109 harboring plasmid pPF8A   PF15BK: Bacillus subtilis harboring plasmid pPF15BK              DN1885   PF11BgK: Bacillus subtilis harboring plasmid pPF11BgK              DN1885   FF1: Bacillus subtilis DN harboring plasmid pPFF1              1885   Primer:   Yeast-soluble 57 kDa β-1 from Elscobia xanthine orythica 73/14 , Primers synthesized for the 5 'region of 3-glucanase.   Y = C or T   R = A or T   K = G or T   N = C, T, A or G   It was synthesized according to the known amino acid sequence of β-1,3-glucanase (Ventom and And Asenjo, supra, 1991).   Primers used for amplification of plasmid pDN1777 DNA by PCR.   The double underlined nucleotide sequence is Bacillus stearothermophyte. The underscored nucleotide sequence corresponding to the ras Corresponds to Ningin Oryka LLG109.   Plasmid:   pPFF1: See FIG.   pPF8A: 2.7 kb BamHI-Bam from Elscobia xanthine orlitica LLG109 HI fragment (see FIG. 2).   pDN520: (Diderichsen, B. and Christiansen, L., FEMS Microbiology Le tters 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., Jen . 4315-4321, 1990).   promoter:   Maltogenic alpha-amilla from Bacillus stearothermophilus (Diderichsen et al., Supra, 1988).   Method:   Genomic DNA isolation:   Chromosomal DNA from Elscobia xanthine orythica was purified by Meade et al. Bacteri ol., 149 (1), p. 114-122, 1982.   Partial live genomic DNA for Elscovia Xanthin Oryka LLG109 Rally building:   Partial chromosomal DNA from Elscovia xanthine Orythica LLG109 with BamHI Digested. The digested DNA was fractionated by agarose gel electrophoresis. About 1.6 A fragment having a size of ４4 kb was isolated and purified. The resulting BamHI fragment was converted to BamHI Digestion-Bacterial alkaline dephosphorylated pUC18 plasmid vector (from Pharmacia) PUC18 BamHI / BAP) and bound to T4 ligase (BRL). This bond mixture Electrocompetent E. coli JM by electroporation using 109 cells were transformed (Bio-Rad Gene Pulser; 125 μFD capacitance, 200 Ω resistance). E. coli JM109 cells were transferred from Sambrook et al., “Molecular Clonlng: A Laborat. ory Manual ”, Cold Spring Harbor Laboratory Press, New York, 1989. No, it was competent.   Elscovia xanthine oligos using radiolabeled DS140 / DS143 PCR products Screening of a partial DNA library from Chika LLG109:   A partial DNA library from the LLG109 strain was spread on a total of 11 plates (approximately With 50 white (recombinant) colonies / plate). Sambrook, supra, 1989. As described, this library was prepared using a sterile nylon membrane (Hybond-N, Amer). sham). Sambrook, 1989. Probed with radiolabeled PCR product: hybridization temperature: 65 ℃, hybridization solution (100ml) 6 × SSC, 0.5% non-fat dry Dry milk. Hybridization was performed overnight (18 hours). Hybridization After the filtration, the filters were washed as follows: 200 ml of 2 at 65 ° C. 10 min + 15 min in x SSC, and 5 min in 0.2 x SSC, 0.1% SDS at 65 ° C. H The filter was subjected to autoradiography (Fuji Film) at -70 ° C overnight. On the filter³²P-labeled probe was detected.   PCR ( Polymerase chain reaction) amplification:   PCR amplification from Elscovia xanthine orichika LLG109   PCR reactions were performed using 100 pmol of each degenerate primer / reaction. 100ng Of Elscobia DNA was used for the reaction. Under conditions recommended by the manufacturer , Taq DNA polymerase (from Promega) was used. Cycling conditions are as follows Hot start: 95 ° C., 1 minute, 40 cycles.   For subsequent sequencing, see Kanungo and Pandy, Biotechniques, 14 (6), p. PCR products were cloned in pUC18 / Smal as described in 912, 1993. I did it.   DNA Gel electrophoresis and Southern blotting:   Agarose gel electrophoresis and Southern blot, according to Sambrook et al., Supra, 1989. Plasmids, restriction endonuclease fragments were analyzed using the setting.   DNA was recovered from agarose gel according to Sambrook et al., 1989, supra.   Radiolabeling of double-stranded DNA fragments:   The double-stranded DNA fragment was radiolabeled according to Sambrook et al., 1989, supra.   DNA Hybridization experiments:   Nylon according to the method described in Sambrook et al., Supra, 1989. Southern blots on filters (Hybond-N, Amersham)³²P-labeled probe And hybridization. Hybridization conditions are as follows Was: hybridization temperature: 65 ° C. Hybridization solution: 6 × SSC, 0.5% non-fat dry milk. Hybridization was performed overnight.   Transformation of E. coli:   Bio-Rad (BIO-RAD) Gene Pulser for Electroporation Device E. coli cells were electroporated as described in the manual. Was transformed.   Transformation of Bacillus subtilis:   Yasbin, R.A. E., Wilson, G .; A. and Young, F .; E., J. Bacteriol., Vol. 12 1, p. 296-304, 1975. Prepare competent cells and transform The quality has changed.   Identification of positive clones:   Identification was performed according to Sambrook et al., Supra, 1989.   DNA Sequencing:   Using the same SEQUENASE sequencing kit from USBI according to the manufacturer's recommendations The DNA sequence was sequenced. 7-death dGTP and sequenase (also US By using a reagent kit for DNA sequencing (using BI) The DNA compact was separated.   Isolation of plasmid DNA:   E. coli: Promega Protocols and Applicatio according to Sambrook et al., Supra, 1989. n E. coli plasmid isolation was performed using the method described in the Guide. .   Bacillus subtilis: Kieser, T., Plasmid, 12, p. 19-36, 1984 The isolation of Bacillus subtilis was performed as described.   Immunological cross-reactivity:   Determination of immunological cross-reactivity using purified β-1,3-glucanase Can be prepared. More specifically, the β of the present invention The -1,3-glucanase antiserum was prepared according to the method described in the following literature. It can be generated by immunizing herons (or other rodents): N. Axelsen et al., A Manual Quantitative Immunoelectrophresis, Blackwell Scie ntific Publications, 1973, Chapter 23, or Johnstone and R.C. Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, 1982 For further details, see p. 27-31). Purified immunoglobulin is obtained from antiserum, for example, Salt precipitation ((NH_Four) SO_Four) Followed by dialysis and ion exchange chromatography, for example , By DEAE-Sephadex ion exchange chromatography, Obtainable. Outcherlony double diffusion analysis (O. Outcher lony, Handbook of Experimental Immunology (Edited by DM Weir), Blackwell Sc ientific Publications, 1967, p. 655-706), cross immunoelectrophoresis (N. Axelsen et al. , Supra, Chapters 3 and 4), or rocket immunoelectrophoresis (N. Axelsen et al., (Chapter 2), immunochemical characterization of the protein was performed.   Culture medium:   LB Agar:   Bacto-tryptophan 10g / l   Pact yeast extract 5g / l   NaCl 10g / l   Pact agar 15g / l   The pH was adjusted to 7.5 with NaOH.   All were autoclaved at 121 ° C. for 30 minutes.   LBGP Agar:   LB agar containing 10 mM potassium phosphate, pH 7.0, 0.4% glucose.   All were autoclaved at 121 ° C. for 30 minutes.   Jacm -7-medium:   Maltodextrin 02 40g   (Average chain length = 12)   20g sucrose   MgSO_Four・ 7H_TwoO 0.64g   KH_TwoPO_Four                              10g   Na_TwoHPO_Four・ 2H_TwoO 10g   Mikrosoy 10ml   Tap water 1 liter   The pH was adjusted to 7.0 with NaOH.   After autoclaving as a concentrated solution, nitrogen source, Casitone and And (NH_Four) HPO_FourWere added separately and the final concentrations were as follows:   Cassiton 9.6g / l   (NH_Four)_TwoHPO_Four                         10.8g / l   All were autoclaved at 121 ° C for 60 minutes.   Fermentation broth-1:   Cassiton 152g   KH_TwoPO_Four                          5.16g   Na_TwoHPO_Four・ 2H_TwoO 5.12g   K_TwoSO_Four                           1.09g   MgCl_Two・ 6H_TwoO 1.11g   Lic trace metal 20ml   Antifoam SB2121 1ml   (StruKtol SB2121, Schill & Seilacher GmbH, Hamburg, Germany)   The volume was adjusted to 1.0 liter with tap water. pH to H_TwoPO_FourAfter adjusting to 5.0 with Sterilized in autoclave at 121 ° C. for 60 minutes.   Sucrose feed solution:Contents in 1 liter flask   MgCl_Two・ 6H_TwoO 8.08g   Lic trace metal 34ml   Antifoam SB2121 1.3ml   241g sucrose   Amount to make tap water 500ml   Autoclave treatment was performed at 121 ° C. for 60 minutes.   CAL 18 -2 medium:   Yeast extract 40.0g   MgSO_Four                              1.3g   Maltodextrin 02 50.0g   NaH_TwoPO_Four                           20.0g   Trace metal 1 6.67ml   Deionized water 1 liter   The pH was adjusted to 6.0 with NaOH and autoclaved at 121 ° C for 60 minutes.   Trace metal solution   Micro Soy:                      mg / l   MnSO_Four・ H_TwoO 500   FeSO_Four・ 7H_TwoO 2000   CuSO_Four・ 5H_TwoO 200   ZnCl_Two                               200   Na_TwoB_FourO₇・ 10H_TwoO 1000   BaCl_Two・ 2H_TwoO 100   (NH_Four)₆Mo₇O_{twenty four}・ 4H_TwoO 100   Na₈−citrat ・ H_TwoO 10000   All were autoclaved at 121 ° C for 60 minutes.   Micro Soy:                      mg / l   MnSO_Four・ H_TwoO 500   FeSO_Four・ 7H_TwoO 2000   CuSO_Four・ 5H_TwoO 200   ZnCl_Two                               200   Na_TwoB_FourO₇・ 10H_TwoO 1000   BaCl_Two・ 2H_TwoO 100   (NH_Four)₆Mo₇O_{twenty four}・ 4H_TwoO 100   Na₈−citrat ・ H_TwoO 10000   All were autoclaved at 121 ° C for 60 minutes.Trace metal 1:   MnSO_Four                             4.48g   FeCl₈・ 6H_TwoO 3.33g   CuSO_Four・ 5H_TwoO 0.625g   ZnSO_Four・ 7H_TwoO 7.12g   Na_TwoMoO_Four                           2.0g   KI 1.0g   Deionized water 1 liter   Lic Trace metals:                    mg / l   MnSO_Four・ H_TwoO 500   FeSO_Four・ 7H_TwoO 2000   CuSO_Four・ 5H_TwoO 200   ZnCl_Two                               200   All were autoclaved at 121 ° C for 60 minutes.   enzyme:   Natural β-1,3-glucana from Elscovia xanthine orythica LLG109 (Ventom and Asenjo, Enzyme Microb. Technol., 13, p. 71, 1991).   Substrate:   AZCL-Kurdaran: Megazyme, Sydney, Australia.   Laminarin: Sigma.   Enzyme assays:   Assay conditions:   Shaking water bath: 1 hour at 37 ° C.   Incubation mixture: 0.5 ml of supernatant + 0.5 ml of 1% w / v AZCL-cle Darane + 0.5 ml acetate buffer (pH 4.0). Also, Kurudaran acetate is relaxed. Dissolved in the buffer. The reaction was stopped with 3 ml of stop reagent. Far away insoluble Kurdalan Rotated by a core machine, the dissolved blue part is 590nm, 1cm by Hitachi spectrophotometer Measured.   Stop reagent:   40 g of sodium acetate   4 g of zinc acetate   Amount to make 200 ml of deionized water   After adjusting the pH to 5.0, it was mixed with 800 ml of 2-methoxyethanol.   Example   Example 1 PCR amplification of natural genomic DNA from Elscovia xanthine Oryka LLG109   Natural genomic DNA from Elscovia xanthine Orythica LLG109 was described above. And amplified by PCR technique.   Elscovia xanthine orichika LLG109 starting from putative start codon? 5 'region of the yeast soluble 57 kDa β-1,3-glucanase gene and For the 3 'region just downstream from the constant stop codon, primers DS96 and DS97 was synthesized. The bases for the EcoRI and BamHI sequences were replaced with 5 'primers (DS 96) and 3 'primer (DS97).   Escovia xanthine orlitica LLG109 strain can be obtained using these primers. When PCR amplification was performed from these genomic DNAs, a 1.7 kb DNA fragment was obtained. PCR fragment Digestion at various restriction sites internal to the gene The pieces were inspected.   Example 2 26 kDa dissolved β-1,3-glu from Elscovia xanthine Orythica LLG109 Determination of partial amino acid sequence from canase   From the N-terminus of the native β-1,3-glucanase and from the native enzyme The following amino acid sequence was initially obtained from the peptide generated by syn digestion.   Standard procedures for proteolytic digestion, peptide separation, and amino acid sequencing Performed according to the law.   N-terminal natural enzyme: N-Ala-Pro-Gly-Asp-Leu-Leu-Trp-Xaa-As p-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 PCR Of chromosomal DNA of Elscovia xanthine Orytica LLG109 by PCR   Synthesized based on back translation of amino acid sequence from β-1,3-glucanase Elsco by PCR using different combinations of mixed oligonucleotides Chromosomal DNA from Via Xanthin Oryka LLG109 was amplified.   Primer DS140 (antisense translation of amino acid sequence FVDGQQF from peptide 2) ) And DS143 (antisense translation of amino acid sequence DYVRVY from peptide 3) The PCR reaction yielded a 180 bp DNA fragment. The PCR product is subsequently Cloned into C18, transformed into E. coli JM109, The leotide sequence was determined (see SEQ ID NO: 3).   Example 4 Southern blot of chromosomal DNA from LLG109 strain using DS140 / DS143 as probe Hybridization analysis   Radiolabeled DS140 / DS143 PCR products as probes under stringent conditions To analyze chromosomal DNA from strain LLG109 by Southern blotting Was. From the BamHI-BamHI band of approximately 2.65 kb or the KpnI-KpnI band of approximately 8 kb. Or strong positive signal from the 1.5 kb BamHI-KpnI band.   In contrast, Southern blot hybridization of chromosomal DNA from 73/14 strain In the analysis, the PCR probes were 8 kb BamHI-BamHI band and 8 kb KpnI-  Strong hybridization to KpnI band or BamHI-KpnI band of about 5 kb (Under the same conditions). In both cases, the radiolabeled probe Southern blots from both Elscobia strains showing strong hybridization In, a band pattern was observed using the PCR products DS133 / DS135 Different from the ones.   Therefore, both LLG109 strains of 73 and 73 / Fourteen strains probably had at least two different β-1,3-glucanase genes.   Example 5 26 kDa β-1 from Elscobia xanthine orlitica LLG109 in Escherichia coli Of the 3,3-glucanase gene   2.7 kb BamH hybridizing to DS140 / DS143 PCR-probe In order to clone the I fragment, an appropriate size (2.7 kb) from the chromosome of the LLG109 strain was used. A DNA fragment of BamHI (± 0.5 kb) was purified and ligated to the BamHI site of pUC18. E. coli JM109 was transformed by electroporation with the conjugate and the same DS 140 / DS143 PCR-Colony Hybridization Using Probes and Conditions Screened.   Isolate clones from a partial gene bank containing the recombinant plasmid pPF8A did. This plasmid was mapped by restriction analysis.   DS140 / DS143 PCR-location and insertion of regions of the plasmid homologous to the probe Was determined by Southern blotting. Plasmid pPF8A has a 2.7 kb BamHI insert was included. Plasmi In the probe, the probe is a 1.5 kb Ba contained within a 2.7 kb BamHI insert. It hybridized to the mHI-KpnI fragment. This fact is due to DS140 / DS14 Results of genomic Southern blot hybridization using 3PCR-probe And matched.   Example 6 Nucleotide sequence of β-1,3-glucanase gene   The nucleotide sequence of the 1.5 kb BamHI-KpnI fragment from pPF8A was Determined by the chain termination method. Double-stranded DNA was denatured by alkali or heat treatment Later, it was used as a template for the sequencing reaction. This region from pPF8A Both strands were sequences according to the indicated sequence strategy.   The complete nucleotide sequence of the 1.5 kb BamHI-KpnI fragment and the predicted amino acid sequence. The noic acid sequence is shown in SEQ ID NO: 1 below.   The nucleotide sequence encodes a protein composed of 306 amino acids. Contains a one nucleotide open reading frame (ORF). Natural tongue Identical or very similar to the amino acid sequence present in the protein The amino acid sequence was found along with the amino acid sequence predicted from the DNA sequence ( The different amino acid residues will be discussed later). In this ORF, the codon ATG Is used as the start codon. The putative ribosome binding site (RBS) It is 4 nucleotides upstream of the don. Upstream of this ORF, two other infrastructures There are lame ATG codons (nucleotides 321 and 351). However, this Since there is no putative RBS at the previous expected position, neither of these Does not seem to function as a start codon. Distribution upstream from coding region Computer scan of rows finds sequences compatible with E. coli-type promoters could not. 3 'downstream from the stop codon TGA 13 base pairs in the G + C rich region (69% G + C) in the flanking region There are inverted repeats of This arrangement can create a stem-loop structure, and And possibly function as a transcription termination signal. 1.5 kb BamHI-KpnI DNA fragment has a G + C content of 67%, and the G + C content of the ORF was previously reported. (70-75% G + C) similar to that of isolated Elscobia chromosomal DNA (Stacke brandt and Prauser, System. Appl. Microbiol., 14 p. 261-265, 1991).   Amino acid sequence of precursor of β-1,3-glucanase   β-1,3-glucanase gene ORF encodes a 306 amino acid protein I do. NH of deduced amino acid sequence_TwoEnds are inside other secreted proteins The characteristics of the found signal sequence are shown (von Heine, Nucl. Acids Res., 14 (11), p. 4683, 1986). After the start codon, three positively charged amino acid residues (histo Gins 5 and arginines 12 and 13) are present, followed by hydrophobic amino acids Long stretch exists (Leu¹⁷−Ala^{twenty three}And Ala²⁶−Ala³⁵). Prokaryotic secretory signa Computer predictions for the sequence The positions were given: i) Potential cleavage sites at positions 35 and 36. From fermentation broth of LLG109 strain Β-1,3-glucanase purified by Ventom and Asenjo, 1991, supra. Natural mature form of NH_TwoThe terminal sequence is APGLLWSDEFDGAAGS It has been determined. This amino acid sequence has the position Thr⁶⁴−Ser⁸⁰Nucleotide sequence in Shows high similarity to what is predicted from:TPESLAWSDEFDGA Indicates AGS. From the predicted amino acid sequence and the natural β-1,3-glucanase Amino acid differences observed between the obtained sequences (see FIG. 3) (underlined ORFs found in pPF8A differ Β-1,3-glucanases, ie, Ventom and Asenjo, supra, 1991. It is suggested that isoenzymes for the listed enzymes can be encoded. After all, Amino acid residue Thr⁶⁴Is NH of isoenzyme of β-1,3-glucanase_TwoForm ends. Therefore, a peptide containing 63 amino acids (Met¹−Val⁶³) As pre-pro area You have to think. Mature β-glucanase (Thr⁶⁴−Gln²⁴³) Has a molecular weight of 26.278 Calculated for the characterized β-1,3-glucanase. Previously calculated from SDS-PAGE by Ventom and Asenjo, supra, 1991. Similar. The predicted pI for this novel enzyme is 3.77, which was previously reported. (Ventom and Asenjo, supra, 1991), which is much lower than the 5.0 pI, It is clear that a new hitherto unknown β-glucanase has been discovered.   Example 7 Cloning of a 26 kDa β-1,3-glucanase gene in Bacillus subtilis And expression   A 1.5 kb BamHI-KpnI fragment from pPF8A was ligated into Bacillus subtilis DN1885. Bacillus stear producing plasmid pPF15BK on plasmid pDN2801 Maltogenic α-amylase from Rothermophilus (Diderichsen, supra , 1990) (Fig. 4). 1.5kb BamH The 1.1 kb BglII-KpnI fragment contained in the I-KpnI fragment was subjected to the same method. Cloning yielded pPF11BgK (FIG. 5). However, Basilah Plasmid transformed into S. subtilis DN1885 (Yasbin et al., J. Bacteriol). . 12., 296-304, 1975) on an LBGP agar plate containing AZCL-kuldran, Alternatively, detectable β- in liquid culture supernatant (CAL 18-2, 3 days, 30 ° C, 250 rpm) It did not generate 1,3-glucanase activity. In addition, the natural expression signal By replacing the well-expressed Bacillus stearothermophilus gene Thus, expression of the β-glucanase gene was performed. Bacillus stearothermophy The promoter of the maltogenic alpha-amylase gene from Las, RBS And signal peptide nucleotide sequences and Thr64 codons and Complete fusion of the remainder of the ORF of β-1,3-glucanase, including the minator Was. This final construct pPFF1 was made as follows: 0.2 kb from pPF11BgK Replace the HindIII-AvaI fragment with the 0.15 kb HindIII-AvaI fragment generated by PCR did. The PCR fragment was obtained from Bacillus stearothermophilus maltogenic α- It contained the RBS of amylase and the coding region of the signal peptide. Plastic PDN520 (Diderichsen et al., Supra, 1988) DNA amplification using immersion kits DK15 and DK16. Depending on the width, PCR fragments were obtained. Plasmid pPFF1 is finally added to Bacillus subtilis DN18 85 (Yasbin et al., Supra, 1975). In this case, at 37 ° C After 24 hours on an LB agar plate containing laminarin (0.04%) By red staining, β-1,3-glucanase activity could be detected.   Finally, pPFF1 in the host lacking protease Bacillus subtilis ToC46 Was transformed.   Example 8 Fermentation of β-1,3-glucanase from Bacillus subtilis DN1885 / pPFF1 and And production   Magnetically coupled stirrer drive, pH and temperature control and carbon source at fixed speed Bacillus subtilis in a 2 liter fermenter equipped with a peristaltic pump that feeds Strain DN1885 / pPFF1 was grown. Of a stock culture of the DN1885 / pPFF1 strain at -80 ° C. Using 100 μl, add 100 ml of Jacm-7-broth and 1 mg of chloramphenicol Contains Inoculate a 500 ml shake flask and bring to 250 rpm and 30 ° C on a rotating table. And grown for 24 hours. This culture (pH = 7.3 and OD₆₀₀= 22) using , A fermenter already containing 1.0 liter fermentation broth 1 was inoculated.   Biomass growth and enzyme production   Prior to inoculation of the culture as described above, 32 g of the sucrose solution was added to the fermentor. Was. Also, sterile filtered air is passed through the lower drive at a rate of 1 liter / minute. It was sparged into the fermentor. Stirring speed was initially set at 500 rpm, but 20% DOT ( The dissolved oxygen tension signal was automatically linked to the set point of dissolved oxygen tension). Peristalsis Using a Watson-Marlow pump, remove after 10 hours of growth. The sucrose feed solution at a constant rate of 7.1 g / hr. The stirrer was rotated near the maximum value and DOT was set to near zero. So, after 19 hours, The sucrose feed rate was reduced to about 6.0 g / h, and after about 82 hours of fermentation time, The feed rate was maintained until empty. After 93 hours, stop fermentation and cool to 4 ° C And stored for later recovery.   The temperature is controlled at 34 ° C. ± 0.1 ° C., and the origin of the dilution of ammonia in water is 5 v / v% H_ThreePO_FourWas added to control the pH 6.00 ± 0.10.   Samples were taken daily, pH, dry weight of biomass and β-1,3-glucanase. Activity was determined as described in the Materials and Methods section. Enzyme concentration in culture supernatant It was determined by hydrolysis of AZCL-Culdran.   Table 1 shows the results of the fermentation.   Example 9 CAL 18 -2 Growth on medium and expression of enzyme   At 30 ° C on LB agar plates containing 10 μg / ml chloramphenicol Bacillus subtilis DN1885 / pPFF1 was grown for 3 days. 100ml CAL 18- 2 medium and 1 mg chloramphenicol into a 500 ml shake flask One colony was transferred. Grow on a rotary table at 250 RPM for 3 days at 30 ° C. Continued, taking samples daily, pH, OD₆₀₀And the dry weight of biomass was determined. Activity determinations were performed as described in the Materials and Methods section, except that 0.05 ml Stop using 2 ml of stop reagent using supernatant + 0.25 ml of Kurdlan + 0.2 ml of extraction Was. Incubation heating was also extended to 20 hours.   Table 2 shows the results of the fermentation.   Example 10 Determination of N-terminal amino acid sequence and purified recombinant β-1,3-glucaner Mass spectrometry   The fermentation broth containing the recombinant β-1,3-glucanase was subjected to ultrafiltration and 20 mM in a Filtron concentrator equipped with a Minisette 10 kDa membrane Was washed with Tris-HCl, pH 8.5. The sample was equilibrated with Tris-HCl, pH 8.5. Applied on a column, and β-1,3-glucanase was added to 10 column volumes of 0-1. Eluted with a linear gradient of M NaCl. Fraction containing β-1,3-glucanase activity Was pooled. Make pool 1.7M with respect to ammonium sulfate and adjust pH to 6.5 did. F equilibrated with 30 mM MOPS, pH 6.5, containing 1.7 M ammonium sulfate. The pool was further purified on a phenyl-Sepharose column. 10 column volume straight line Of β-1,3-glucanase with 10 mM Tris-HCl, pH 8.5 using a gradient of did. Fractions containing β-1,3-glucanase activity were pooled and 10 mM boric acid Extensive dialysis against sodium pH 9.0. The dialyzed sample was treated with 10 mM sodium borate. Β-1,3-glucanase applied on a Mono Q column equilibrated with Eluted with a volume of 0-1 M NaCl.   N-terminal amino acid of purified recombinant β-1,3-glucanase The acid sequence is identical to the deduced N-terminal sequence from SEQ ID NO: 1.   Matrix for Flight Mass Spectrometry of Purified Recombinant β-1,3-Glucanase Auxiliary laser desorption ionization time gives a mass of 26,462 Da, which is within experimental error According to the mass calculated from the amino acid sequence in the box.   Abbreviation   amino acid   A = Ala = alanine   V = Val = Valine   L = Leu = leucine   I = Ile = isoleucine   P = Pro = proline   F = Phe = phenylalanine   W = Trp = Tryptophan   M = Met = methionine   G = Gly = glycine   S = Ser = Serine   T = Thr = Threonine   C = Cys = cysteine   Y = Tyr = Tyrosine   N = Asn = asparagine   Q = Gln = Glutamine   D = Asp = aspartic acid   E = Glu = glutamic acid   K = Lys = Lysine   R = Arg = arginine   H = His = histidine   Nucleic acid base   A = Adenine   G = guanine   C = cytosine   T = thymine (only in DNA)   U = uracil (only for RNA)

[Procedure for Amendment] Article 184-8, Paragraph 1 of the Patent Act [Date of Submission] January 13, 1997 [Contents of Amendment] Claims 1. A DNA construct comprising a DNA sequence that encodes an enzyme that exhibits beta-1,3-glucanase activity and is capable of opening a cell wall by permeating the cell wall, said DNA sequence comprising: a) a sequence comprising: Comprising the DNA sequence shown in SEQ ID NO: 1, or b) comprising an analog of the DNA sequence shown in SEQ ID NO: 1, said analog comprising: i) an oligonucleotide probe identical to the DNA sequence shown in SEQ ID NO: 1 And ii) encodes a polypeptide that is at least 70% homologous to the polypeptide encoded by the DNA sequence comprising the DNA sequence set forth in SEQ ID NO: 1, or iii) Elscobia xanthine orytica LLG109 Antibodies raised against purified beta-1,3-glucanase encoded by the DNA sequence shown in SEQ ID NO: 1 derived from Encodes a polypeptide that is epidemiologically reactive, DNA construct. 2. The DNA construct according to claim 1, comprising a DNA sequence encoding an enzyme exhibiting beta-1,3-glucanase activity, wherein the DNA sequence comprises the DNA sequence shown in SEQ ID NO: 1. 3. A DNA construct according to claims 1 and 2, wherein the DNA sequence comprises the DNA sequence shown in SEQ ID NO: 3. 4. The DNA construct according to any one of claims 1 to 3, wherein the DNA sequence is derived from a microorganism. 5. The DNA construct according to claim 4, wherein the DNA sequence is derived from a filamentous fungus or yeast. 6. The DNA construct according to claim 5, wherein the DNA sequence is derived from the group consisting of Aspergillus, Trichoderma, Fusarium or Humicola. 7. The DNA construct of claim 4, wherein the DNA sequence is derived from a bacterium. 8. The DNA sequence is derived from a bacterium from Bacillus, in particular Bacillus liheniformis, Bacillus amyloliquefaciens, Bacillus circulans, or Clostridium, especially Clostridium termorum, or Elscobia, Arthrobacter, Cytofaga or Rhodotermus. The DNA construct according to claim 7, which is a DNA construct. 9. 9. A DNA construct according to claim 8, wherein the DNA sequence is derived from Elscovia xanthine orlitica, in particular LSG109 (DSM No. 10297). Ten. A recombinant expression vector comprising the DNA construct according to any one of claims 1 to 9. 11． 11. The recombinant expression vector according to claim 10, wherein said vector comprises a Bacillus stearothermophilus maltogenic alpha-amylase promoter. 12． 12. The recombinant expression vector according to claims 10 and 11, wherein said vector comprises a Bacillus stearothermophilus maltogenic alpha-amylase signal. 13. 13. The recombinant expression vector according to any one of claims 10 to 12, comprising a sequence from the pPFF1 plasmid. 14. A cell comprising the DNA construct according to any one of claims 1 to 9 or the recombinant expression vector according to any one of claims 10 to 13. 15． 15. The cell according to claim 14, wherein the cell is a microbial cell. 16． 16. The cell according to claim 15, wherein the cell is a bacterial cell. 17． The bacterium is a bacterium, such as a strain of Elscovia, Arthrobacter, Cytofaga, Rhodotermus, or Bacillus, such as Bacillus subtilis, Bacillus ligeniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus. ), A strain of Bacillus alcalophilus Bacillus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lactus, Bacillus megaterium or Bacillus slingensis, or Streptomyces, for example, Streptomyces 17. The cell of claim 16, which is a bacterial cell selected from the group consisting of a strain of Rividans or Streptomyces murinus, or a strain of Escherichia, for example, E. coli. 18． 18. The cell according to claims 16 and 17, wherein the bacterial cell is Bacillus subtilis DN1885. 19. 18. The cell according to claims 16 and 17, wherein the bacterial cell is Bacillus subtilis ToC46. 20. 16. The cell of claim 14, wherein the cell is a bacterial cell different from the cell from which the DNA sequence is derived. twenty one. 16. The cell according to claim 14, wherein the cell is a fungal cell. twenty two. 22. The cell of claim 21, wherein the fungal cell is a fungal cell selected from the group of strains of Aspergillus, Trichoderma, Fusarium, or Humicola. twenty three. 16. The cell of claim 14, wherein the cell is a fungal cell different from the cell from which the DNA sequence was derived. twenty four. 24. Culturing the cell according to any one of claims 14 to 23 under conditions enabling production of an enzyme exhibiting beta-1,3-glucanase activity, and recovering the enzyme from the culture. A method for producing said enzyme, comprising: twenty five. A novel enzyme exhibiting beta-1,3-glucanase activity, which is a) encoded by the DNA construct of any one of claims 1 to 9, and b) produced by the method of claim 24. 26. 26. The antibody is immunologically reactive with antibodies raised against purified beta-1,3-glucanase encoded by the DNA sequence shown in SEQ ID NO: 1 derived from Elscobia xanthine orlitica LLG109. Novel enzyme according to 1. 27. 27. The novel enzyme of claims 25 and 26, wherein said enzyme has an apparent molecular weight of about 26 kDa. 28. 28. The novel enzyme according to any one of claims 25 to 27, wherein said enzyme has a pI in the range of about 3.5-4. 29. An enzyme preparation useful for modifying or degrading a substance containing beta-glucan, comprising the novel enzyme according to any one of claims 25 to 28. 30. 30. The enzyme preparation of claim 29, comprising one or more other cell wall degrading enzymes selected from the group of enzymes having cellulolytic, mannose, chitin or proteolytic activity. 31. 31. The enzyme of claim 30, wherein the enzyme is selected from the group consisting of endoglucanases, e.g., cellulases and beta-1,6-glucanases, and mannanases, endo- or exo-chitinases, proteases, and alpha- or beta-mannosidases. Enzymes. 32. The enzyme preparation according to claims 30 and 31, comprising 200L, an enzyme from the group comprising Cellulase, Cellulase CP and Cellulase CT, and / or Chitinase. 33. The enzyme according to any one of claims 25 to 28, wherein the enzyme or the enzyme preparation according to any one of claims 29 to 32 is used for modifying or degrading a substance containing beta-glucan. Use of. 34. 34. Use according to claim 33, wherein the enzyme or enzyme preparation is used in the preparation of protoplasts. 35. 34. Use of an enzyme according to claim 33, wherein the enzyme or enzyme preparation is used in the production of pigments, colorants, flavors, yeast extracts. 36. 34. Use of an enzyme according to claim 33, wherein the enzyme or enzyme preparation is used in the manufacture of a formulation. 37. 34. Use of an enzyme according to claim 33, wherein the enzyme or enzyme preparation is used in a formulation.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/21 C12R 1: 125) (C12N 9/24 C12R 1: 125) (81) Designated country EP (AT, BE, CH) , DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR) , NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AL, AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, MK, MN, MW MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, US, UZ, VN (72) Inventor Hedegaard, Lisbeth Denmark, Decor-2880 Baksba Elt Novo Are, Novo Nordisk Actiesel Skab (no address) (72) Inventor Harquier Torven, Denmark, Denmark-2880 Baksba Elt Novo Are, Novo Nordisk Actiesel Sukab (no address) (72) Inventors Asenjo, Juan A. La Reina Las Perdisses, Santiago, Chile, 0277-See (72) Inventors Sabah, Demitris United Kingdom, Reading Earl G6 2, Wyjay, Northburn Claus 17

Claims (1)

[Claims] 1. A DNA construct comprising a DNA sequence encoding an enzyme exhibiting β-1,3-glucanase activity, said DNA sequence comprising: a) the DNA sequence shown in SEQ ID NO: 1, or b) the sequence Comprising an analog of the DNA sequence shown in SEQ ID NO: 1; i) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID NO: 1; and ii) converts the DNA sequence shown in SEQ ID NO: 1 Encoding a polypeptide homologous to the polypeptide encoded by the DNA sequence comprising, or iii) purified β encoded by the DNA sequence shown in SEQ ID NO: 1 derived from Elscovia xanthine orlitica LLG109 A DNA construct encoding a polypeptide that is immunologically reactive with antibodies raised against 1,3-glucanase. 2. The DNA construct according to claim 1, comprising a DNA sequence encoding an enzyme exhibiting β-1,3-glucanase activity, wherein the DNA sequence comprises the DNA sequence shown in SEQ ID NO: 1. 3. A DNA construct according to claims 1 and 2, wherein the DNA sequence comprises the DNA sequence shown in SEQ ID NO: 3. 4. The DNA construct according to any one of claims 1 to 3, wherein the DNA sequence is derived from a microorganism. 5. The DNA construct according to claim 4, wherein the DNA sequence is derived from a filamentous fungus or yeast. 6. The DNA construct according to claim 5, wherein the DNA sequence is derived from the group consisting of Aspergillus, Trichoderma, Fusarium or Humicola. 7. The DNA construct of claim 4, wherein the DNA sequence is derived from a bacterium. 8. The DNA sequence is derived from a bacterium from Bacillus, in particular Bacillus liheniformis, Bacillus amyloliquefaciens, Bacillus circulans, or Clostridium, especially Clostridium termorum, or Elscobia, Arthrobacter, Cytofaga or Rhodotermus. The DNA construct according to claim 7, which is a DNA construct. 9. 9. A DNA construct according to claim 8, wherein the DNA sequence is derived from Elscovia xanthine orlitica, in particular LSG109 (DSM No. 10297). Ten. A recombinant expression vector comprising the DNA construct according to any one of claims 1 to 9. 11． 11. The recombinant expression vector according to claim 10, wherein the vector comprises a Bacillus stearothermophilus maltogenic α-amylase promoter. 12． 12. The recombinant expression vector according to claims 10 and 11, wherein said vector comprises a Bacillus stearothermophilus maltogenic α-amylase signal. 13. The recombinant expression vector according to any one of claims 10 to 12, comprising a sequence from the pPFF1 plasmid. 14. A cell comprising the DNA construct according to any one of claims 1 to 9 or the recombinant expression vector according to any one of claims 10 to 13. 15． 15. The cell according to claim 14, wherein the cell is a microbial cell. 16． 16. The cell according to claim 15, wherein the cell is a bacterial cell. 17． The bacterium is a bacterium, such as a strain of Elscobia, Arthrobacter, Cytofaga, Rhodotermus, or Bacillus, such as Bacillus subtilis, Bacillus ligeniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus al. Bacillus bacillus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lactus, a strain of Bacillus megaterium or Bacillus slingensis, or Streptomyces, such as Streptomyces lividans or Streptomyces 17. The cell according to claim 16, which is a bacterial cell selected from the group consisting of a strain of Ses murinus or a strain of Escherichia, for example E. coli. 18． 18. The cell according to claims 16 and 17, wherein the bacterial cell is Bacillus subtilis DN1885. 19. 18. The cell according to claims 16 and 17, wherein the bacterial cell is Bacillus subtilis ToC46. 20. 16. The cell of claim 14, wherein the cell is a bacterial cell different from the cell from which the DNA sequence is derived. twenty one. 16. The cell according to claim 14, wherein the cell is a fungal cell. twenty two. 22. The cell of claim 21, wherein the fungal cell is a fungal cell selected from the group of strains of Aspergillus, Trichoderma, Fusarium, or Humicola. twenty three. 16. The cell of claim 14, wherein the cell is a fungal cell different from the cell from which the DNA sequence was derived. twenty four. 24. Culturing the cell according to any one of claims 14 to 23 under conditions enabling production of an enzyme exhibiting β-1,3-glucanase activity, and recovering the enzyme from the culture. A method for producing said enzyme, comprising: twenty five. A novel enzyme exhibiting β-1,3-glucanase activity, which is a) encoded by the DNA construct of any one of claims 1 to 9, and b) produced by the method of claim 24. 26. 26. The antibody is immunologically reactive with an antibody raised against a purified β-1,3-glucanase encoded by the DNA sequence shown in SEQ ID NO: 1 derived from Elscobia xanthine orlitica LLG109. Novel enzyme according to 1. 27. 27. The novel enzyme of claims 25 and 26, wherein said enzyme has an apparent molecular weight of about 26 kDa. 28. 28. The novel enzyme according to any one of claims 25 to 27, wherein said enzyme has a pI in the range of about 3.5-4. 29. An enzyme preparation useful for modifying or degrading a substance containing β-glucan, comprising the novel enzyme according to any one of claims 25 to 28. 30. 30. The enzyme preparation of claim 29, comprising one or more other cell wall degrading enzymes selected from the group of enzymes having cellulolytic, mannose, chitin or proteolytic activity. 31. 31. The enzyme of claim 30, wherein the enzyme is selected from the group consisting of endoglucanases, e.g., cellulases and beta-1,6-glucanases, and mannanases, endo- or exo-chitinases, proteases, and alpha- or beta-mannosidases. Enzymes. 32. The enzyme preparation according to claims 30 and 31, comprising 200L, an enzyme from the group comprising Cellulase, Cellulase CP and Cellulase CT, and / or Chitinase. 33. The enzyme according to any one of claims 25 to 28, wherein the enzyme or the enzyme preparation according to any one of claims 29 to 32 is used for modifying or degrading a substance containing β-glucan. Use of. 34. 34. Use according to claim 33, wherein the enzyme or enzyme preparation is used in the preparation of protoplasts. 35. 34. Use of an enzyme according to claim 33, wherein the enzyme or enzyme preparation is used in the production of pigments, colorants, flavors, yeast extracts. 36. 34. Use of an enzyme according to claim 33, wherein the enzyme or enzyme preparation is used in the manufacture of a formulation. 37. 34. Use of an enzyme according to claim 33, wherein the enzyme or enzyme preparation is used in a formulation.