DNA constructs and methods of producing cellulytic enzymes

An isolated nucleic acid constructs encoding cellulytic enzymes derived from a strain of Bacillus agaradherens, recombinant vectors and host cells comprising such constructs, and methods for obtaining cellulytic enzymes.

FIELD OF THE INVENTION
 The present invention relates to isolated nucleic acid sequences and
 constructs encoding cellulytic enzymes derived from a strain of Bacillus,
 recombinant expression vectors and host cells comprising such constructs,
 and methods for obtaining cellulytic enzymes.
 BACKGROUND OF THE INVENTION
 PCT publication WO 94/01532 describes a new species of alkalophilic
 Bacillus, initially named Bacillus sp. AC13, as well as proteases,
 xylanases and cellulases obtainable therefrom. A sample of the strain was
 deposited as NCIMB 40482. WO 94/01532 also describes methods for the
 production of these enzymes by cultivation of a strain of Bacillus sp.
 AC13. However, WO 94/01532 does not describe nucleic acid constructs
 comprising a nucleic acid sequence encoding cellulytic enzymes derived
 from a strain Bacillus sp. AC13, or methods of producing these cellulytic
 enzymes by recombinant DNA technology.
 The same new species as described in WO 94/01532 has been described by
 Nielsen et al. (1995) Microbiology 141:1745-1761, with the now established
 name, Bacillus agaradhierens. A sample of the strain has been deposited as
 DSM 8721. Nielsen et al. (1995) supra, however, do not describe nucleic
 acid sequences or constructs encoding cellulytic enzymes derived from a
 strain Bacillus agaradhierens, or methods of producing these cellulytic
 enzymes by recombinant DNA technology.
 SUMMARY OF THE INVENTION
 The invention features an isolated DNA sequence derived from Bacillus
 encoding a cellulytic enzyme, thereby making it possible to prepare a
 mono-component enzyme preparation.
 Accordingly, in one aspect, the invention provides an isolated DNA sequence
 derived from Bacillus agaradhierens encoding a polypeptide having
 cellulytic enzyme activity. In a specific embodiment, the isolated DNA
 sequence is the sequence of SEQ ID NO:1. In another related embodiment,
 the isolated DNA sequence is a DNA sequence encoding a cellulytic enzyme
 having more than 98% homology to the cellulytic enzyme encoded by the DNA
 sequence of SEQ ID NO:1. Included in the invention is an isolated DNA
 sequence complementary to SEQ ID NO:1, and a fragment of the sequence of
 SEQ ID NO:1 (or its complementary sequence) that is at least 15 base pairs
 in length that selectively hybridizes under stringent conditions to DNA
 sequences encoding the cellulytic enzyme of SEQ ID NO:1. In still further
 embodiments, the DNA sequence is isolated from a Bacillus strain
 identified by the deposit accession number DSM 8721 or NCIMB 40482.
 In further aspects, the invention provides a DNA construct having the DNA
 sequence of SEQ ID NO:1, an expression vector harboring the DNA construct
 of the invention, a cell having the DNA construct or expression vector of
 the invention, as well as a method of producing a cellulytic enzyme by
 culturing the cell of the invention under conditions permitting the
 production of the cellulytic enzyme, and recovering the cellulytic enzyme
 from the culture.
 In another aspect, the invention features an isolated polypeptide encoded
 by SEQ ID NO:1 and having cellulytic activity. The invention includes an
 isolated polypeptide having the amino acid sequence of SEQ ID NO:2, or a
 polypeptide having an amino acid sequence with at least 80%, 90%, or 95%
 identity with the amino acid sequence of SEQ ID NO:2.
 The invention further features an enzyme preparation comprising the
 cellulytic enzyme encoded by the DNA sequence of SEQ ID NO:1.
 DETAILED DISCLOSURE OF THE INVENTION
 Before the methods and compositions of the present invention are described
 and disclosed it is to be understood that this invention is not limited to
 the particular methods and compositions described as such may, of course,
 vary. It is also to be understood that the terminology used herein is for
 the purpose of describing particular embodiments only, and is not intended
 to be limiting since the scope of the present invention will be limited
 only by the appended claims.
 It must be noted that as used in this specification and the appended
 claims, the singular forms "a", "an" and "the" include plural referents
 unless the context clearly dictates otherwise. Thus, for example,
 reference to "a DNA sequence" includes a plurality of DNA sequences and
 different types of DNA sequences.
 Unless defined otherwise all technical and scientific terms used herein
 have the same meaning as commonly understood by one of ordinary skill in
 the art to which this invention belongs. Although any materials or methods
 similar or equivalent to those described herein can be used in the
 practice or testing of the present invention, the preferred methods and
 materials are now described. All publications mentioned herein are
 incorporated herein by reference for the purpose of describing and
 disclosing the particular information for which the publication was cited.
 The publications discussed above are provided solely for their disclosure
 prior to the filing date of the present application. Nothing herein is to
 be construed as an admission that the inventor is not entitled to antedate
 such disclosure by virtue of prior invention.
 Isolated DNA Sequences and DNA Constructs
 The present invention provides an isolated DNA sequence and a construct
 comprising the DNA sequence encoding a cellulytic enzyme. The DNA sequence
 of the invention includes (a) the DNA sequence of SEQ ID NO:1, (b) a DNA
 sequence encoding a polypeptide having more than 98% homology with the
 cellulytic enzyme encoded by SEQ ID NO:1, (c) a sequence complementary to
 SEQ ID NO:1, and (d) a fragment of the sequence of (a), (b), or (c) that
 is at least 15 base pairs in length that selectively hybridizes under
 stringent conditions to DNA sequences encoding the cellulytic enzyme of
 SEQ ID NO:1.
 As defined herein the term "DNA construct" is intended to indicate any
 nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin.
 The term "construct" is intended to indicate a nucleic acid segment which
 may be single- or double-stranded, and which may be based on a complete or
 partial naturally occurring nucleotide sequence encoding a cellulytic
 enzyme of interest. It is understood that such nucleotide sequences
 include intentionally manipulated nucleotide sequences, e.g., subjected to
 site-directed mutagenesis, and sequences that are degenerate as a result
 of the genetic code. All degenerate nucleotide sequences are included in
 the invention so long as the cellulytic enzyme encoded by the nucleotide
 sequence is functionally unchanged. The construct may optionally contain
 other nucleic acid segments.
 The DNA construct of the invention preferably is of microbial origin,
 preferably derived from a strain of Bacillus. In its most preferred
 embodiments, the DNA construct of the invention is derived from a strain
 of the new alkalophilic species Bacillus agaradhierens, formerly referred
 to as Bacillus AC13.
 The DNA construct of the invention encoding the cellulytic enzyme may
 suitably be of genomic or cDNA origin, for instance obtained by preparing
 a genomic or cDNA library and screening for DNA sequences coding for all
 or part of the cellulytic enzyme by hybridization using synthetic
 oligonucleotide probes in accordance with standard techniques (cf. e.g.
 Sambrook et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring
 Harbor, N.Y.).
 The nucleic acid construct of the invention encoding the cellulytic enzyme
 may also be prepared synthetically by established standard methods, e.g.
 the phosphoamidite method described by Beaucage and Caruthers (1981)
 Tetrahedron Letters 22:1859-1869, or the method described by Matthes et
 al. (1984) EMBO J. 3:801-805. According to the phosphoamidite method,
 oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer,
 purified, annealed, ligated and cloned in suitable vectors.
 The nucleic acid construct may also be prepared by polymerase chain
 reaction using specific primers, for instance as described in U.S. Pat.
 No. 4,683,202 or by Saiki et al. (1988) Science 239:487-491.
 Furthermore, the nucleic acid construct may be of mixed synthetic and
 genomic DNA, mixed synthetic and cDNA, or mixed genomic and cDNA origin
 prepared by ligating fragments of synthetic, genomic or cDNA origin (as
 appropriate), the fragments corresponding to various parts of the entire
 nucleic acid construct, in accordance with standard techniques.
 The present invention also relates to polynucleotides which are capable of
 hybridizing under high stringency conditions with an oligonucleotide probe
 which hybridizes under the same conditions with the nucleic acid sequence
 set forth in SEQ ID NO:1 or its complementary strand (Sambrook et al.
 (1989) Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring
 Harbor, N.Y.). Hybridization indicates that the analogous nucleic acid
 sequence hybridizes to the oligonucleotide probe corresponding to the
 polypeptide encoding part of the nucleic acid sequence of SEQ ID NO:1,
 under low to high stringency conditions (for example, prehybridization and
 hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 mg/ml
 sheared and denatured salmon sperm DNA, and either 50, 35 or 25% formamide
 for high, medium and low stringencies, respectively), following standard
 Southern blotting procedures.
 SEQ ID NO:1 may be used to identify and clone DNA encoding a cellulytic
 enzyme from other strains of different genera or species according to
 methods well known in the art. Thus, genomic or cDNA library prepared from
 such other organisms may be screened for DNA which hybridizes with SEQ ID
 NO:1 and encodes a cellulytic enzyme. Genomic or other DNA from such other
 organisms may be separated by agarose or polyacrylamide gel
 electrophoresis, or other separation techniques. DNA from the libraries or
 the separated DNA may be transferred to and immobilized on nitrocellulose
 or other suitable carrier material. In order to identify clones or DNA
 which is homologous with SEQ ID NO:1, the carrier material is used in a
 Southern blot in which the carrier material is finally washed three times
 for 30 minutes each using 2.times.SSC, 0.2% SDS at preferably not higher
 than 50.degree. C., more preferably not higher than 55.degree. C., more
 preferably not higher than 60.degree. C., more preferably not higher than
 65.degree. C., even more preferably not higher than 70.degree. C.,
 especially not higher than 75.degree. C. Molecules to which the
 oligonucleotide probe hybridizes under these conditions are detected using
 a X-ray film.
 An analogous DNA sequence may preferably be isolated from a strain of
 Bacillus, preferably a strain of Bacillus agaradhierens, on the basis of
 the DNA sequence presented as SEQ ID NO:1, or any fragment thereof, e.g.
 using the procedures described herein, and thus, e.g. be an allelic or
 species variant of the DNA sequence comprising the DNA sequence presented
 herein.
 Alternatively, the analogous sequence may be constructed on the basis of
 the DNA sequence presented as SEQ ID NO:1, or any fragment thereof, e.g.
 by introduction of nucleotide substitutions which do not give rise to
 another amino acid sequence of the cellulytic enzyme encoded by the DNA
 sequence, but which corresponds to the codon usage of the host organism
 intended for production of the enzyme, or by introduction of nucleotide
 substitutions which may give rise to a different amino acid sequence.
 When carrying out nucleotide substitutions, amino acid changes are
 preferably of a minor nature, that is conservative amino acid
 substitutions that do not significantly affect the folding or activity of
 the protein, small deletions, typically of one to about 30 amino acids;
 small amino- or carboxyl-terminal extensions, such as an amino-terminal
 methionine residue, a small linker peptide of up to about 20-25 residues,
 or a small extension that facilitates purification, such as a
 poly-histidine tract, an antigenic epitope or a binding domain. Examples
 of conservative substitutions are within the group of basic amino acids
 (such as arginine, lysine, histidine), acidic amino acids (such as
 glutamic acid and aspartic acid), polar amino acids (such as glutamine and
 asparagine), hydrophobic amino acids (such as leucine, isoleucine,
 valine), aromatic amino acids (such as phenylalanine, tryptophan,
 tyrosine) and small amino acids (such as glycine, alanine, serine,
 threonine, methionine). For a general description of nucleotide
 substitution, see e.g. Ford et al. (1991) Protein Expression and
 Purification 2:95-107.
 It will be apparent to persons skilled in the art that such substitutions
 can be made outside the regions critical to the function of the molecule
 and still result in an active cellulytic enzyme. Amino acids essential to
 the activity of the cellulase encoded by the DNA construct of the
 invention, and therefore preferably not subject to substitution, may be
 identified according to procedures known in the art, such as site-directed
 mutagenesis or alanine-scanning mutagenesis (cf. e.g. Cunningham and Wells
 (1989) Science 244:1081-1085). In the latter technique mutations are
 introduced at every residue in the molecule, and the resultant mutant
 molecules are tested for biological (i.e. proteolytic) activity to
 identify amino acid residues that are critical to the activity of the
 molecule. Sites of substrate-enzyme interaction can also be determined by
 analysis of crystal structure as determined by such techniques as nuclear
 magnetic resonance analysis, crystallography or photoaffinity labelling
 (cf. e.g. de Vos et al. (1992) Science 255:306-312; Smith et al. (1992) J.
 Mol. Biol. 224:899-904; Wlodaver et al. (1992) FEBS Lett. 309:59-64).
 Typically, the analogous DNA sequence is highly homologous to the DNA
 sequence, such is more than 98% homologous to the DNA sequence presented
 as SEQ ID NO:1 encoding a cellulytic enzyme, preferably at least 99%
 homologous to said DNA sequence.
 The degree of homology referred to above is determined as the degree of
 identity between the two sequences indicating a derivation of the first
 sequence from the second. The degree of identity between two nucleic acid
 sequences may be determined by means of computer programs known in the art
 such as GAP provided in the GCG program package (Needleman and Wunsch
 (1970) Journal of Molecular Biology 48:443-453). For purposes of
 determining the degree of identity between two nucleic acid sequences for
 the present invention, GAP is used with the following settings: GAP
 creation penalty of 5.0 and GAP extension penalty of 0.3.
 The DNA sequence encoding the cellulytic enzyme may be isolated by
 conventional methods. The techniques used to isolate or clone a nucleic
 acid sequence encoding a polypeptide are known in the art and include
 isolation from genomic DNA, preparation from cDNA, or a combination
 thereof. The cloning of the nucleic sequences of the present invention
 from such genomic DNA can be effected, e.g., by using the well known
 polymerase chain reaction (PCR). See, e.g., Innis et al. (1990) A Guide to
 Methods and Application, Academic Press, New York. The nucleic acid
 sequence may be cloned from a strain of the Bacillus agaradhierens, e.g.
 the strain DSM 8721 or the strain NCIMB 40482, producing the polypeptide,
 or another or related organism and thus, for example, may be an allelic or
 species variant of the polypeptide encoding region of the nucleic acid
 sequence.
 The term "isolated" nucleic acid sequence as used herein refers to a
 nucleic acid sequence which is essentially free of other nucleci acid
 sequences, e.g., at least about 20% pure, preferably at least about 40%
 pure, more preferably about 60% pure, even more preferably about 80% pure,
 most preferably about 90% pure, and even most preferably about 95% pure,
 as determined by agarose gel electrophoresis. For example, an isolated
 nucleic acid sequence can be obtained by standard cloning procedures used
 in genetic engineering to relocate the nucleic acid sequence from its
 natural location to a different site where it will be reproduced. The
 cloning procedures may involve excision and isolation of a desired nucleic
 acid fragment comprising the nucleic acid sequence encoding the
 polypeptide, insertion of the fragment into a vector molecule, and
 incorporation of the recombinant vector into a host cell where multiple
 copies or clones of the nucleic acid sequence will be replicated. The
 nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,
 synthetic origin, or any combinations thereof.
 Microbial Sources
 The DNA construct of the invention preferably is of microbial origin,
 preferably derived from a strain of Bacillus. In a more preferred
 embodiment, the DNA construct of the invention is derived from a strain of
 the new species Bacillus agaradhierens. As described above, Bacillus
 agaradhierens is a new species of alkalophilic Bacilli, which has been
 disclosed by Nielsen et al. (1995) supra. The strain was formerly referred
 to as Bacillus AC13. Therefore, in another embodiment, the DNA construct
 of the invention is derived from a strain of Bacillus AC13.
 The type strain of Bacillus agaradhierens is the strain DSM 8721, which
 strain has been deposited in the open collection of Deutsche Sammlung von
 Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, DE-3300
 Braunschweig, Germany.
 The strain Bacillus AC13, also a representative of the new species Bacillus
 agaradhierens, has been deposited according to the Budapest Treaty on the
 International Recognition of the Deposit of Microorganisms for the
 Purposes of Patent Procedure at National Collections of Industrial and
 Marine Bacteria, Ltd. (NCIB), 23 St. Machar Drive, GB-Aberdeen AB2 1RY,
 United Kingdom, on Mar. 3, 1992 and allotted the deposit number NCIB
 40482. As an International Depository Authority under the Budapest Treaty,
 NCIB 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 Commissioner 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 deposit fulfills the
 requirements of European patent applications relating to micro-organisms
 according to Rule 28 EPC.
 In a more preferred embodiment, the DNA construct of the invention is
 derived from the strain NCIMB 40482, or the strain DSM 8721, or mutants or
 variants thereof. The DNA sequence encoding the cellulytic enzyme may be
 isolated from these deposits by standard methods, e.g. as described in
 Example 1.
 Further, said DNA sequence may be isolated by screening a cDNA library of a
 strain of Bacillus agaradhierens, followed by selection for clones
 expressing the cellulytic enzyme (e.g. as defined by their ability to
 degrade cellulose). The appropriate DNA sequence may then be isolated from
 the clone by standard procedures.
 Alternatively, the DNA encoding the cellulytic enzyme may, in accordance
 with well-known procedures, conveniently be isolated from DNA from the
 source in question by use of synthetic oligonucleotide probes prepared on
 the basis of a DNA sequence disclosed herein. For instance, a suitable
 oligonucleotide probe may be prepared on the basis of the nucleotide
 sequences presented as SEQ ID NO:1, or any suitable fragment thereof.
 Recombinant Expression Vectors
 In another aspect, the invention provides a recombinant expression vector
 comprising the DNA construct of the invention.
 The expression vector of the invention may be any expression vector that is
 conveniently subjected to recombinant DNA procedures, and the choice of
 vector will often depend on the host cell into which it is to be
 introduced. Thus, the vector may be an autonomously replicating vector,
 i.e. a vector which exists as an extrachromosomal entity, the replication
 of which is independent of chromosomal replication, e.g. a plasmid.
 Alternatively, the vector may be one which, when introduced into a host
 cell, is integrated into the host cell genome and replicated together with
 the chromosome(s) into which it has been integrated.
 In the expression vector of the invention, the DNA sequence encoding the
 cellulytic enzyme preferably is operably linked to additional segments
 required for transcription of the DNA. In general, the expression vector
 is derived from plasmid or viral DNA, or may contain elements of both. The
 term, "operably linked" indicates that the segments are arranged so that
 they function in concert for their intended purposes, e.g. transcription
 initiates in a promoter and proceeds through the DNA sequence coding for
 the cellulytic enzyme.
 Thus, in the expression vector of the invention, the DNA sequence encoding
 the cellulytic enzyme preferably should be operably connected to a
 suitable promoter and terminator sequence. The promoter may be any DNA
 sequence which shows transcriptional activity in the host cell of choice
 and may be derived from genes encoding proteins either homologous or
 heterologous to the host cell. The procedures used to ligate the DNA
 sequences coding for the cellulytic enzyme, the promoter and the
 terminator, respectively, and to insert them into suitable vectors are
 well known to persons skilled in the art (cf., for instance, Sambrook et
 al.(1989) supra.
 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. Examples of suitable
 promoters for directing the transcription of the DNA encoding the
 cellulytic enzyme of the invention in bacterial host cells include the
 promoter of the Bacillus stearothermophilis maltogenic amylase gene, the
 Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens
 BAN amylase gene, the Bacillus subtilis alkaline protease gene, or the
 Bacillus pumilus xylanase or xylosidase gene, the phage Lambda P.sub.R or
 P.sub.L promoters, or the E. coli lac, trp or tac promoters.
 Examples of suitable promoters for use in yeast host cells include
 promoters from yeast glycolytic genes (Hitzeman et al. (1980) J. Biol.
 Chem. 255:12073-12080; Alber and Kawasaki (1982) J. Mol. Appl. Gen.
 1:419-434) or alcohol dehydrogenase genes (Young et al. (1982) in Genetic
 Engineering of Microorganisms for Chemicals (Hollaender et al, eds.),
 Plenum Press, New York), or the TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4c
 (Russell et al. (1983) Nature 304:652-654) promoters.
 Examples of suitable promoters for use in filamentous fungus host cells
 are, for instance, the ADH3 promoter (McKnight et al. (1985) EMBO J.
 4:2093-2099) or the tpiA promoter. Examples of other useful promoters are
 those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor
 miehei aspartic proteinase, A. niger neutral a-amylase, A. niger acid
 stable a-amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor
 miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate
 isomerase or A. nidulans acetamidase. Preferred are the TAKA-amylase and
 gluA promoters.
 The expression vector of the invention may further comprise a DNA sequence
 enabling the vector to replicate in the host cell in question. The
 expression vector may also comprise a selectable marker, e.g. a gene the
 product of which complements a defect in the host cell, such as the gene
 coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe
 TPI gene (described by Russell (1985) Gene 40:125-130), or one which
 confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,
 chloramphenicol, neomycin, hygromycin or methotrexate. For filamentous
 fungi, selectable markers include amdS, pyrG, argB, niaD and sC.
 To direct the cellulytic enzyme into the secretory pathway of the host
 cells, a secretory signal sequence (also known as a leader sequence,
 prepro sequence or pre sequence) may be provided in the expression vector.
 The secretory signal sequence is joined to the DNA sequence encoding the
 cellulytic enzyme in the correct reading frame. Secretory signal sequences
 are commonly positioned 5' to the DNA sequence encoding the cellulytic
 enzyme. The secretory signal sequence may be that normally associated with
 the cellulytic enzyme or may be from a gene encoding another secreted
 protein.
 In a preferred embodiment, the expression vector of the invention may
 comprise a secretory signal sequence substantially identical to the
 secretory signal encoding sequence of the Bacillus licheniformis a-amylase
 gene, e.g. as described in WO 86/05812.
 Also, measures for amplification of the expression may be taken, e.g. by
 tandem amplification techniques, involving single or double crossing-over,
 or by multicopy techniques, e.g. as described in U.S. Pat. No. 4,959,316
 or WO 91/09129. Alternatively the expression vector may include a
 temperature sensitive origin of replication, e.g. as described in EP
 283,075.
 Procedures for ligating DNA sequences encoding the cellulytic enzyme, the
 promoter and optionally the terminator and/or secretory signal sequence,
 respectively, and to insert them into suitable vectors containing the
 information necessary for replication, are well known to persons skilled
 in the art (cf., for example, Sambrook et al. (1989) supra.
 Host Cells
 In yet another aspect the invention provides a host cell containing the DNA
 construct of the invention and/or the recombinant expression vector of the
 invention.
 The DNA construct of the invention may be either homologous or heterologous
 to the host in question. If homologous to the host cell, i.e. produced by
 the host cell in nature, it will typically be operably connected to
 another promoter sequence or, if applicable, another secretory signal
 sequence arid/or terminator sequence than in its natural environment. In
 this context, the term "homologous" is intended to include a cDNA sequence
 encoding a cellulytic enzyme native to the host organism in question. The
 term "heterologous" is intended to include a DNA sequence not expressed by
 the host cell in nature. Thus, the DNA sequence may be from another
 organism, or it may be a synthetic sequence.
 The host cell of the invention, into which the DNA construct or the
 recombinant expression vector of the invention is to be introduced, may be
 any cell which is capable of producing the cellulytic enzyme and includes
 bacteria, yeast, fungi and higher eukaryotic cells.
 Examples of bacterial host cells which, on cultivation, are capable of
 producing the cellulytic enzyme of the invention are grampositive bacteria
 such as strains of Bacillus, in particular a strain of B. subtilis, B.
 licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.
 alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus,
 B. megatherium, B. pumilus, B. thuringiensis or B. agaradhierens, or
 strains of Streptomyces, in particular a strain of S. lividans or S.
 murinus, or gramnegative bacteria such as Echerichia 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. (1989) supra).
 When expressing the cellulytic enzyme in bacteria such as E. coli, the
 cellulase 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 cellulytic enzyme is refolded by diluting the denaturing agent.
 In the latter case, the cellulytic enzyme 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
 cellulytic enzyme.
 Examples of suitable yeasts cells include cells of Saccharomyces spp. or
 Schizosaccharomyces spp., in particular strains of Saccharomyces
 cerevisiae or Saccharomyces kluyveri. Methods for transforming yeast cells
 with heterologous DNA and producing heterologous polypeptides therefrom
 are described, e.g. in U.S. Pat. Nos. 4,599,311, 4,931,373, 4,870,008,
 5,037,743, and 4,845,075, all of which are hereby specifically
 incorporated by reference. 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 POT1 vector disclosed in U.S. Pat. No.
 4,931,373. The DNA sequence encoding the cellulytic enzyme 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 Kluyveromyces, such as K. lactis, Hansenula, e.g. H.
 polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al. (1986) J. Gen.
 Microbiol. 132:3459-3465; U.S. Pat. No. 4,882,279).
 Examples of other fungal cells are cells of filamentous fungi, e.g.
 Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in
 particular strains of A. oryzae, A. nidulans or A. niger. The use of
 Aspergillus spp. for the expression of proteins have been described in
 e.g., EP 272,277 and EP 230,023. The transformation of F. oxysporum may,
 for instance, be carried out as described by Malardier et al. (1989) Gene
 78:147-156.
 The transformed or transfected host cell described above is then cultured
 in a suitable nutrient medium under conditions permitting the expression
 of the cellulytic enzyme, after which the resulting cellulytic 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
 cellulytic enzyme 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 cellulytic enzyme in question.
 Method of Producing Cellulytic Enzymes
 The present invention also relates to methods for producing a polypeptide
 of the present invention comprising (a) cultivating a Bacillus strain to
 produce a supernatant comprising the polypeptide; and (b) recovering the
 polypeptide.
 The present invention also relates to methods for producing a polypeptide
 of the present invention comprising (a) cultivating a host cell under
 conditions conducive to expression of the polypeptide; and (b) recovering
 the polypeptide.
 In both methods, the cells are cultivated in a nutrient medium suitable for
 production of the polypeptide using methods known in the art. For example,
 the cell may be cultivated by shake flask cultivation, small-scale or
 large-scale fermentation (including continuous, batch, fed-batch, or solid
 state fermentations) in laboratory or industrial fermentors performed in a
 suitable medium and under conditions allowing the polypeptide to be
 expressed and/or isolated. The cultivation takes place in a suitable
 nutrient medium comprising carbon and nitrogen sources and inorganic
 salts, using procedures known in the art (see, e.g., references for
 bacteria and yeast; Bennett, J. W. and LaSure, L., eds. (1991) More Gene
 Manipulations in Fungi, Academic Press, California). Suitable media are
 available from commercial suppliers or may be prepared according to
 published compositions (e.g., in catalogues of the American Type Culture
 Collection). If the polypeptide is secreted into the nutrient medium, the
 polypeptide can be recovered directly from the medium. If the polypeptide
 is not secreted, it is recovered from cell lysates.
 The polypeptides may be detected using methods known in the art that are
 specific for the polypeptides. These detection methods may include use of
 specific antibodies, formation of an enzyme product, or disappearance of
 an enzyme substrate. For example, an enzyme assay may be used to determine
 the activity of the polypeptide. Procedures for determining cellulytic
 activity are known in the art and are described in the examples below.
 The resulting polypeptide may be recovered by methods known in the art. For
 example, the polypeptide may be recovered from the nutrient medium by
 conventional procedures including, but not limited to, centrifugation,
 filtration, extraction, spray-drying, evaporation, or precipitation. The
 recovered polypeptide may then be further purified by a variety of
 chromatographic procedures, e.g., ion exchange chromatography, gel
 filtration chromatography, affinity chromatography, or the like.
 The polypeptides of the present invention may be purified by a variety of
 procedures known in the art including, but not limited to, chromatography
 (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size
 exclusion), electrophoretic procedures (e.g., preparative isoelectric
 focusing (IEF), differential solubility (e.g., ammonium sulfate
 precipitation), or extraction (see, e.g., Protein Purification (Janson and
 Ryden, eds.), VCH Publishers, New York, 1989).
 Polypeptide Preparations
 In a still further aspect, the present invention relates to polypeptide
 compositions and preparations which are enriched in the cellulytic enzyme
 of the invention encoded by a DNA construct of the invention, or produced
 by the method of the invention.
 The enzyme preparation of the invention may be one which comprises the
 polypeptide of the invention as the major enzymatic component, and may in
 particular be a mono-component enzyme preparation. Alternatively, the
 composition may comprise multiple enzymatic activities, such as an
 aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a
 catalase, a cellulase, a chitinase, a cutinase, a deoxyribonuclease, an is
 esterase, an alpha-galactosidase, a beta-galactosidase, a glucoamylase, an
 alpha-glucosidase, a beta-glucosidase, a haloperoxidase, an invertase, a
 laccase, a lipase, a mannosidase, a mutanase, an oxidase, a pectinolytic
 enzyme, a peroxidase, a phytase, a polyphenoloxidase, a proteolytic
 enzyme, a ribonuclease, or a xylanase. The additional enzyme(s) may be
 producible by means of a microorganism belonging to the genus Aspergillus,
 preferably Aspergillus niger, Aspergillus aculeatus, Aspergillus awamori
 or Aspergillus oryzae, or Trichoderma, Humicola, preferably Humicola
 insolens, or Fusarium, preferably Fusarium graminearum.
 The polypeptide compositions may be prepared in accordance with methods
 known in the art and may be in the form of a liquid or a dry composition.
 For instance, the polypeptide composition may be in the form of a
 granulate or a microgranulate. The polypeptide to be included in the
 composition may be stabilized in accordance with methods known in the art.
 Examples are given below of preferred uses of the polypeptide compositions
 of the invention. The dosage of the polypeptide 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.
 Uses
 The enzyme preparation according to the invention may be applied in
 industrial processes conventionally involving the action of cellulytic
 enzymes. Major applications for cellulytic enzymes are found in the
 detergent industry, in the textile industry, in paper pulp processing
 industry, and in the food and feed industry.
 In preferred embodiments the enzyme preparation of the invention may be
 used for degradation or modification of plant material, e.g. cell walls,
 for the treatment of fabric or textile, preferably for preventing
 backstaining, for bio-polishing or "stone-washing" cellulosic fabric, in
 the treatment of paper pulp, preferably for debarking, defibration, fibre
 modification, enzymatic de-inking or drainage improvement.

EXAMPLES
 The following examples are put forth so as to provide those of ordinary
 skill in the art with a complete disclosure and description of how to make
 and use various constructs and perform the various methods of the present
 invention and are not intended to limit the is scope of what the inventors
 regard as their invention. Unless indicated otherwise, parts are parts by
 weight, temperature is in degrees centigrade, and pressure is at or near
 atmospheric pressure. Efforts have been made to ensure accuracy with
 respect to numbers used (e.g., length of DNA sequences, molecular weights,
 amounts, particular components, etc.), but some deviations should be
 accounted for.
 EXAMPLE 1
 Materials and Methods
 Cellulytic Activity. Cellulytic activity may be measured in cellulase
 viscosity units (CEVU), determined at pH 9.0 with carboxymethyl cellulose
 (CMC) as substrate.
 Cellulase viscosity units are determined relatively to an enzyme standard
 (&lt;1% water, kept in N.sub.2 atmosphere at -20.degree. C.; arch standard at
 -80.degree. C.). The standard used, 17-1187, is 4400 CEVU/g under standard
 incubation conditions, i.e., pH 9.0, Tris Buffer 0.1 M, CMC Hercules 7 LFD
 substrate 33.3 g/l, 40.0.degree. C. for 30 minutes.
 Donor Organism. Bacillus AC13 NCIMB 40482 (identical to Bacillus
 agaradhierens DSM 8721) expressing the endoglucanase enzyme encoding the
 DNA sequence of SEQ ID NO:1.
 Other Strains. E. coli strain: Cells of E. coli SJ2 (Diderichsen et al.
 (1990) J. Bacteriol. 172:4315-4321), which encodes alpha-acetolactate
 decarboxylase, an exoenzyme from Bacillus brevis were prepared for and
 transformed by electroporation using a Gene Pulser.TM. electroporator from
 BIO-RAD as described by the supplier.
 Bacillus subtilis PL2304: This strain is the B. subtilis DN1885
 (Diderichsen et al. (1990) supra), disrupted in the transcriptional unit
 of the known Bacillus subtilis cellulase gene, resulting in cellulase
 negative cells. The disruption was performed essentially as described by
 Hoch & Losick (1993) in Bacillus subtilis and other Gram-Positive Bacteria
 (Sonenshein, A. L., ed.), pp. 618).
 Plasmids. pSJ1678: Described in WO 94/19454; pDN1981: Described by
 Jorgensen et al. (1990) Gene 96:37-41).
 EXAMPLE 2
 Cloning of Bacillus agaradhierens Endoglucanase Gene
 Genomic DNA Preparation. The strain NCIMB 40482 (identical to Bacillus
 agaradhierens DSM 8721) was propagated in liquid medium as described in WO
 94/01532. After 16 hours of incubation at 30.degree. C. and 300 rpm, the
 cells were harvested, and genomic DNA was isolated by the method described
 by Pitcher et al. (1989) Lett. Appl. Microbiol. 8:151-156).
 Genomic Library Construction. Genomic DNA was partially digested with
 restriction enzyme Sau3A and size-fractionated by electrophoresis on a
 0.7% agarose gel. Fragments of between 2 and 7 kb in size were isolated by
 electrophoresis onto DEAE-cellulose paper (Dretzen et al. (1981) Anal.
 Biochem. 112:295-298). Isolated DNA fragments were ligated to BamHI
 digested, pSJ1678 plasmid DNA.
 PCR Amplification. In order to obtain the endoglucanse gene as ligated to
 the pSJ1678 vector, the ligation mixture was used as DNA template in a PCR
 reaction containing 200 mM of each nucleotide (dATP, dCTP, dGTP and dTTP),
 2.5 mM MgCl.sub.2, Expand High Fidelity buffer, 2.0 units of Expand High
 Fidelity PCR system enzyme mix and 300 nM of each of the following
 primers: Primer 1 (#9555):
 5'-TCACAGATCCTCGCGAATTGGTGCGGCCGCGTNGTNG-ARGARCAYGGNC-3' (SEQ ID NO:3).
 Primer 1 is a degenerated primer designed to match the amino acid sequence
 (Val-Val-Glu-Glu-His-Gly-Gln) (SEQ ID NO:4) of the N-terminal amino acid
 sequence presented in WO94/01532. The last amino acid is only presented by
 the first nucleotide of the codon namely C. C is the 3'-nucleotide of the
 primer. Furthermore, a NotI site is included at the 5'-end for cloning
 purposes these nucleotides are underlined. Primer 2 (#9029):
 5'-CAGAGCAAGAGATTACGCGC-3' (SEQ ID-NO:5). Primer 2 corresponds to a
 sequence present in the pSJ1678 vector.
 The PCR cycling was performed in a Hans Landgraf THERMOCYCLERO (Hans
 Landgraf, Germany), following the profile:
 1.times.(120 seconds at 94.degree. C.);
 10.times.(10 seconds at 94.degree. C.; 30 seconds at 55.degree. C.; 240
 seconds at 72.degree. C.);
 30.times.(10 seconds at 94.degree. C.; 30 seconds at 55.degree. C.; 180
 seconds at 72.degree. C.; adding 20 seconds to the keep time at 72.degree.
 C. for each new cycle); and
 1.times.(300 seconds at 72.degree. C.).
 The PCR product was gel purified by gel electrophoresis in a 0.7% agarose
 gel, and the relevant fragment (approx. 1.7 kb) was excised from the gel
 and purified using QIAquickO Gel extraction Kit (Qiagen, USA) according to
 the manufacturer's instructions. The purified DNA was eluted in 50 .mu.l
 of 10 mM Tris-HCl, pH 8.5.
 This DNA was used as a template for a PCR reamplification using the same is
 primers, mixture and cycle profile as above.
 The PCR product was gel purified by gel eletrophoresis in a 0.7% agarose
 gel, and the relevant fragment was excised from the gel and purified using
 QIAquick Gel extraction Kit. The purified DNA was eluted in 50 .mu.l of 10
 mM Tris-HCl, pH 8.5.
 The purified DNA was digested with NotI and HindIII, gel purified as above,
 and ligated to the vector pBluescriptII KS- (Stratagene, USA), also
 digested with NotI and HindIII, and the ligation mixture was used to
 transform E. coli SJ2.
 Cells were plated on LB agar plates containing ampicillin (200 .mu.g/ml)
 supplemented with X-gal (5-Bromo-4-chloro-3-indolyl
 alpha-D-Galactopyranoside, 50 .mu.g/ml).
 Identification and Charaterization of Positive Clones. The transformed
 cells were plated on LB agar plates containing ampicillin (200 .mu.g/ml)
 supplemented with X-gal (5-Bromo-4-chloro-3-indolyl
 alpha-D-Galactopyranoside, 50 .mu.g/ml), and incubated at 37.degree. C.
 overnight. The next day white colonies were rescued by restreaking these
 onto fresh LB-ampicillin agar plates and incubated at 37.degree. C.
 overnight. The day after, single colonies of each clone were transferred
 to liquid LB medium containing ampicillin (200 .mu.g/ml), and incubated
 overnight at 37.degree. C. with shaking at 250 rpm.
 Plasmids were extracted from the liquid cultures using QIAgen Plasmid
 Purification mini kit. Five-.mu.l samples of the plasmids are digested
 with NotI and HindIII. The digestions were checked by gel electrophoresis
 on a 0.7% agarose gel (NuSieve, FMC). The appearence of a DNA fragment of
 approximately 1.0 kb indicated a positive clone.
 Nucleotide Sequencing the Cloned DNA Fragment. Qiagen purified plasmid DNA
 was sequenced with the Taq deoxy terminal cycle sequencing kit (Perkin
 Elmer, USA) and the primer "Reverse" or the primer "Forward": Reverse:
 5'-GTTTTCCCAGTCACGAC-3' (SEQ ID NO:6), Forward:
 5'-GCGGATAACAATTTCACACAGG-3' (SEQ ID NO:7).
 The DNA was sequenced using an Applied Biosystems 373A automated sequencer
 according to the manufacturers instructions. Analysis of the sequence data
 is performed according to Devereux et al. (1984) Nucleic Acids Res.
 12:387-395).
 From this sequence new primers could be designed for performing Inverse PCR
 [cf. McPherson et al. (eds) in PCR-A practical approach; 1991 IRL Press).
 Inverse PCR on Genomic DNA of Strain NCIMB 40482. Genomic DNA was isolated
 as described above. 2 mg of pure genomic DNA was digested with EcoRI. The
 EcoRI was heat inactivated at 65.degree. C. for 20 minutes, after which a
 phenol:chloroform extraction of DNA was performed. DNA was finally ethanol
 precipitated and resuspended in 20 ml TE.
 1 ml of EcoRI digested DNA was ligated with T4-DNA ligase in 100 ml
 reaction mixture containing T4 ligase buffer and 1 Unit T4-DNA ligase
 (Boehringer Mannheim, Germany). After 18 hours of ligation at 14.degree.
 C., the ligase was heat inactivated at 68.degree. C. for 10 minutes. In
 order to linearize the circulized genomic DNA fragments prior to Inverse
 PCR, the ligation mixture was supplemented with 10 U of BstEII (a BstEII
 site was present internally of the DNA sequence obtained above).
 50 ml of the BstEII digested ligation mixture was used as template in a PCR
 reaction containing 200 mM of each nucleotide (dATP, dCTP, dGTP and dTTP),
 2.5 mM MgCl.sub.2, Expand High Fidelity buffer, 2.0 units of Expand High
 Fidelity PCR system enzyme mix, and 300 nM of each of the following
 primers:
 Primer 3 (#19719): 5'-TGACCCGTACGGTCCGTGGG-3' (SEQ ID NO:8), and Primer 4
 (#19720): 5'-GGCTCTTGATTTTGTGTCCACC-3' (SEQ ID NO:9).
 The PCR cycling was performed in a Hans Landgraf THERMOCYCLER (Hans
 Landgraf, Germany), following the profile:
 1.times.(120 seconds at 94.degree. C.);
 10.times.(10 seconds at 94.degree. C.; 30 seconds at 55.degree. C.; 240
 seconds at 72.degree. C.);
 30.times.(10 seconds at 94.degree. C.; 30 seconds at 55.degree. C.; 180
 seconds at 72.degree. C. adding 20 seconds to the keep time at 72.degree.
 C. for each new cycle); and
 1.times.(300 seconds at 72.degree. C.).
 The PCR product was gel purified by gel eletrophoresis in a 0.7% agarose
 gel, and the relevant fragment (approx. 4-5 kb) was excised from the gel
 and purified using QIAquick Gel extraction Kit. The purified DNA was
 eluted in 50 .mu.l of 10 mM Tris-HCl, pH 8.5.
 Nucleotide Sequencing the Inverse-PCR DNA Fragment. Qiagen purified DNA was
 sequenced with the Taq deoxy terminal cycle sequencing kit (Perkin Elmer,
 USA), and the primer 1, 3 and 4 described above, using an Applied
 Biosystems 373A automated sequencer according to the manufacturers
 instructions. Analysis of the sequence data is performed according to
 Devereux et al. (1984) supra).
 The entire nucleotide sequence corresponding to the open reading frame of
 the alkaline endoglucanase is presented as SEQ ID NO:1, and the derived
 protein sequence is presented as SEQ ID NO:2.
 Example 3
 Expression of the Alkaline Endoglucanase in Bacillus subtilis
 The nucleotide sequence in SEQ ID NO:1 was cloned by PCR for introduction
 in an expression plasmid pDN1981. PCR was performed as described above on
 500 ng of genomic DNA, using the following two primers, containing NdeI
 and KpnI restriction sites for introducing the endoglucanase encoding DNA
 sequence to pDN1981 for expression:
 Primer 5 (#20887):
 5'-GTAGGCTCAGTCATATGTTACACATTGAAAGGGGAGGAGAATCATGAAAAAGATAACTACTATTTTTGTCG
 -3' (SEQ ID NO:10); and
 Primer 6 (#21318):
 5'-GTACCTCGCGGGTACCAAGCGGCCGCTTAATTGAGTGGTTCCCACGGACCG-3' (SEQ ID NO:11).
 After PCR cycling, the PCR fragment was purified, and the purified DNA was
 eluted in 50 .mu.l of 10 mM Tris-HCl, pH 8.5, digested with NdeI and KpnI,
 purified and ligated to digested pDN1981. The ligation mixture was used to
 transform B. subtilis PL2304. Competent cells were prepared and
 transformed as described by Yasbin et al. (1975) J. Bacteriol.
 121:296-304).
 EXAMPLE 4
 Isolation and Test of Bacillus subtilis Transformants
 The transformed cells were plated on LB agar plates containing 10 mg/ml
 Kanamycin, 0.4% glucose, 10 mM KH2PO4 and 0.1% AZCL HE-cellulose
 (Megazyme, Australia), and incubated at 37.degree. C. for 18 hours.
 Endoglucanase positive colonies were identified as colonies surrounded by
 a blue halo.
 Each of the positive transformants were inoculated in 10 ml TY-medium
 containing 10 mg/ml Kanamycin. After 1 day of incubation at 37.degree. C.
 and stirring at 250 rpm, 50 ml supernatant was removed. The endoglucanase
 activity was identified by adding 50 ml supernatant to holes punched in
 the agar of LB agar plates containing 0.1% AZCL HE-cellulose.
 After 16 hours of incubation at 37.degree. C., blue halos surrounding holes
 indicated expression of the endoglucanase in Bacillus subtilis.
 EXAMPLE 5
 Characterization of the Purified Enzyme
 The cellulytic enzyme consists of a signal peptide (i.e. amino acid
 residues 1 to 26), a catalytic core domain belonging to the family 5 (1)
 of the Bacillus subfamily (i.e. amino acid residues 1 to 306), followed by
 a short linker region (i.e. amino residues 307 to 328), and finally new
 class of cellulose binding domain, CBD (i.e. amino acid residues 329 to
 376).
 The molar extinction coefficient was determined as 114,000. The molecular
 weight was approximately 43 kD. It was determined that the enzyme does not
 contain a cysteine residue, and the charged amino acids give a calculated
 pI of around 4.
 The enzyme has a broad pH profile and very high alkaline activity, and has
 a temperature optima of around 60.degree. C. The product is fully stable
 after one hour of incubation in an American standard detergent solution at
 40.degree. C.
 The purified enzyme has a maximal activity at 60.degree. C., 3 times higher
 than that observed at 40.degree. C.
 EXAMPLE 6
 Expression of the Alkaline Endoglucanase in Bacillus subtilis
 The nucleotide sequence in SEQ ID NO: 12 was cloned by PCR for introduction
 in an expression plasmid pDN1981.
 PCR was performed as described below on 500 ng of genomic DNA, using the
 following two primers containing NdeI and KpnI (the KpnI site is
 conveniently present in the amplified sequence) restriction sites for
 introducing the endoglucanase encoding DNA sequence to pDN1981 for
 expression:
 Primer 5 (#20887): 5'-GTA GGC TCA GTC ATA TGT TAC ACA TTG AAA GGG GAG GAG
 AAT CAT GAA AAA GAT AAC TAC TAT TTT TGT CG-3' (SEQ ID NO:10), and
 Primer 7 (#100084): 5'-CCT CGC GAG GTA CCA GCG GCC GCG TAC CAC CAA TTA AGT
 ATG GTA C-3' (SEQ ID NO: 14)
 The underlined nucleotides of Primer 5 corresponds to the NdeI site, and
 the underlined nucleotides in the Primer 7 is part of the KpnI site
 present in the sequence.
 Using the Expand.TM. Long Template PCR system (available from Boehringer
 Mannheim, Germany) amplification was performed using a mixture consisting
 of (Buffer 1 diluted 10 times) and 200 .mu.M of each dNTP, 2.5 units of
 Enzyme mix (Boehringer Mannheim, Germany) and 500 pmol of each primer.
 The PCR reactions was performed using a DNA Thermal Cycler (available from
 Landgraf, Germany). One incubation at 94.degree. C. for 2 min followed by
 ten cycles of PCR performed using a cycle profile of denaturation at
 94.degree. C. for 10 seconds, annealing at 55.degree. C. for 30 seconds,
 and extension at 68.degree. C. for 4 minutes. Followed by 25 cycles of PCR
 performed using a cycle profile of denaturation at 94.degree. C. for 10
 seconds, annealing at 55.degree. C. for 30 seconds, and extension at
 68.degree. C. for 3 minutes (this duration of extension is extended with
 20 seconds for each of the 25 cycles).
 Aliquots of 10 .mu.l of the amplification product is analysed by
 electrophoresis in 0.7% agarose gels (NuSieve, FMC) with ReadyLoad 100 bp
 DNA ladder (GibcoBRL, Denmark) as a size marker.
 After PCR cycling, the PCR fragment was purified using QIAquick PCR column
 Kit (Qiagen, USA) according to the manufacturer's instructions. The
 purified DNA was eluted in 50 .mu.l of 10 mM Tris-HCl, pH 8.5, digested
 with NdeI and KpnI, and purified and ligated to digested pDN1981. The
 ligation mixture was used to transform B. subtilis PL2304.
 Competent cells were prepared and transformed as described by Yasbin et al.
 [Yasbin R E, Wilson G A & Young F E; Transformation and transfection in
 lysogenic strains of Bacillus subtilis: evidence for selective induction
 of prophage in competent cells; J Bacteriol 1975 121 296-304].
 Isolation and Test of Bacillus subtilis Transformants
 The transformed cells were plated on LB agar plates containing 10 mg/ml
 Kanamycin, 0.4% glucose, 10 mM KH2PO4 and 0.1% AZCL HE-cellulose
 (Megazyme, Australia), and incubated at 37.degree. C. for 18 hours.
 Endoglucanase positive colonies were identified as colonies surrounded by
 a blue halo.
 Each of the positive transformants were inoculated in 10 ml TY-medium
 containing 10 mg/ml Kanamycin. After 1 day of incubation at 37.degree. C.
 and stirring at 250 rpm, 50 ml supernatant was removed. The endoglucanase
 activity was identified by adding 50 ml supernatant to holes punched in
 the agar of LB agar plates containing 0.1% AZCL HE-cellulose.
 After 16 hours of incubation at 37.degree. C., blue halos surrounding holes
 indicated expression of the endoglucanase in Bacillus subtilis.
 EXAMPLE 7
 Analysis of the Cloned Sequence
 The protein sequence derived from the cloned endoglucanase gene shows an
 endoglucanase of the following composition:
 Amino acid residues 1 to 26 correspond to a signal peptide; amino acid
 residues 27 to 326 constitute the actual endoglucanase (homologues to
 other family 5 glycosyl hydrolases); amino acid residues 327 to 354
 correspond to a linker; amino acid residues 355 to 400 correspond to a
 cellulose binding domain (as described in Example 2); amino acid residues
 401 to 416 correspond to a linker; and amino acid residues 417 to 462
 constitute a second cellulose binding domain (highly homologues to the
 first one (at amino acid residues 355 to 400)).
 The molar extinction coefficient was determined as 146,370. The molecular
 weight was approximately 52 kD.
 For the protein without the signal sequence the molar extinction
 coefficient was determined as 146.370. The molecular weight was
 approximately 49 kD.
 The enzyme has no cysteine, and the charged amino acids give a calculated
 pI of around 4.
 SEQUENCE LISTING
 (1) GENERAL INFORMATION:
 (iii) NUMBER OF SEQUENCES: 14
 (2) INFORMATION FOR SEQ ID NO: 1:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 1203 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: Bacillus agaradherens
 (B) STRAIN: AC13
 (ix) FEATURE:
 (A) NAME/KEY: CDS
 (B) LOCATION: 1..1203
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
 ATG AAA AAG ATA ACT ACT ATT TTT GTC GTA TTG CTT ATG ACA GTG GCG 48
 Met Lys Lys Ile Thr Thr Ile Phe Val Val Leu Leu Met Thr Val Ala
 1 5 10 15
 TTG TTC AGT ATA GGA AAC ACG ACT GCT GCT GAT AAT GAT TCA GTT GTA 96
 Leu Phe Ser Ile Gly Asn Thr Thr Ala Ala Asp Asn Asp Ser Val Val
 20 25 30
 GAA GAA CAT GGG CAA TTA AGT ATT AGT AAC GGT GAA TTA GTC AAT GAA 144
 Glu Glu His Gly Gln Leu Ser Ile Ser Asn Gly Glu Leu Val Asn Glu
 35 40 45
 CGA GGC GAA CAA GTT CAG TTA AAA GGG ATG AGT TCC CAT GGT TTG CAA 192
 Arg Gly Glu Gln Val Gln Leu Lys Gly Met Ser Ser His Gly Leu Gln
 50 55 60
 TGG TAC GGT CAA TTT GTA AAC TAT GAA AGT ATG AAA TGG CTA AGA GAT 240
 Trp Tyr Gly Gln Phe Val Asn Tyr Glu Ser Met Lys Trp Leu Arg Asp
 65 70 75 80
 GAT TGG GGA ATA AAT GTA TTC CGA GCA GCA ATG TAT ACC TCT TCA GGA 288
 Asp Trp Gly Ile Asn Val Phe Arg Ala Ala Met Tyr Thr Ser Ser Gly
 85 90 95
 GGA TAT ATT GAT GAT CCA TCA GTA AAG GAA AAA GTA AAA GAG GCT GTT 336
 Gly Tyr Ile Asp Asp Pro Ser Val Lys Glu Lys Val Lys Glu Ala Val
 100 105 110
 GAA GCT GCG ATA GAC CTT GAT ATA TAT GTG ATC ATT GAT TGG CAT ATC 384
 Glu Ala Ala Ile Asp Leu Asp Ile Tyr Val Ile Ile Asp Trp His Ile
 115 120 125
 CTT TCA GAC AAT GAC CCA AAT ATA TAT AAA GAA GAA GCG AAG GAT TTC 432
 Leu Ser Asp Asn Asp Pro Asn Ile Tyr Lys Glu Glu Ala Lys Asp Phe
 130 135 140
 TTT GAT GAA ATG TCA GAG TTG TAT GGA GAC TAT CCG AAT GTG ATA TAC 480
 Phe Asp Glu Met Ser Glu Leu Tyr Gly Asp Tyr Pro Asn Val Ile Tyr
 145 150 155 160
 GAA ATT GCA AAT GAA CCG AAT GGT AGT GAT GTT ACG TGG GGC AAT CAA 528
 Glu Ile Ala Asn Glu Pro Asn Gly Ser Asp Val Thr Trp Gly Asn Gln
 165 170 175
 ATA AAA CCG TAT GCA GAG GAA GTC ATT CCG ATT ATT CGT AAC AAT GAC 576
 Ile Lys Pro Tyr Ala Glu Glu Val Ile Pro Ile Ile Arg Asn Asn Asp
 180 185 190
 CCT AAT AAC ATT ATT ATT GTA GGT ACA GGT ACA TGG AGT CAG GAT GTC 624
 Pro Asn Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser Gln Asp Val
 195 200 205
 CAT CAT GCA GCT GAT AAT CAG CTT GCA GAT CCT AAC GTC ATG TAT GCA 672
 His His Ala Ala Asp Asn Gln Leu Ala Asp Pro Asn Val Met Tyr Ala
 210 215 220
 TTT CAT TTT TAT GCA GGG ACA CAT GGT CAA AAT TTA CGA GAC CAA GTA 720
 Phe His Phe Tyr Ala Gly Thr His Gly Gln Asn Leu Arg Asp Gln Val
 225 230 235 240
 GAT TAT GCA TTA GAT CAA GGA GCA GCG ATA TTT GTT AGT GAA TGG GGA 768
 Asp Tyr Ala Leu Asp Gln Gly Ala Ala Ile Phe Val Ser Glu Trp Gly
 245 250 255
 ACA AGT GCA GCT ACA GGT GAT GGT GGC GTG TTT TTA GAT GAA GCA CAA 816
 Thr Ser Ala Ala Thr Gly Asp Gly Gly Val Phe Leu Asp Glu Ala Gln
 260 265 270
 GTG TGG ATT GAC TTT ATG GAT GAA AGA AAT TTA AGC TGG GCC AAC TGG 864
 Val Trp Ile Asp Phe Met Asp Glu Arg Asn Leu Ser Trp Ala Asn Trp
 275 280 285
 TCT CTA ACG CAT AAA GAT GAG TCA TCT GCA GCG TTA ATG CCA GGT GCA 912
 Ser Leu Thr His Lys Asp Glu Ser Ser Ala Ala Leu Met Pro Gly Ala
 290 295 300
 AAT CCA ACT GGT GGT TGG ACA GAG GCT GAA CTA TCT CCA TCT GGT ACA 960
 Asn Pro Thr Gly Gly Trp Thr Glu Ala Glu Leu Ser Pro Ser Gly Thr
 305 310 315 320
 TTT GTG AGG GAA AAA ATA AGA GAA TCA GCA TCT ATT CCG CCA AGC GAT 1008
 Phe Val Arg Glu Lys Ile Arg Glu Ser Ala Ser Ile Pro Pro Ser Asp
 325 330 335
 CCA ACA CCG CCA TCT GAT CCA GGA GAA CCG GAT CCA ACG CCC CCA AGT 1056
 Pro Thr Pro Pro Ser Asp Pro Gly Glu Pro Asp Pro Thr Pro Pro Ser
 340 345 350
 GAT CCA GGA GAG TAT CCA GCA TGG GAT CCA AAT CAA ATT TAC ACA AAT 1104
 Asp Pro Gly Glu Tyr Pro Ala Trp Asp Pro Asn Gln Ile Tyr Thr Asn
 355 360 365
 GAA ATT GTG TAC CAT AAC GGC CAG CTA TGG CAA GCA AAA TGG TGG ACA 1152
 Glu Ile Val Tyr His Asn Gly Gln Leu Trp Gln Ala Lys Trp Trp Thr
 370 375 380
 CAA AAT CAA GAG CCA GGT GAC CCG TAC GGT CCG TGG GAA CCA CTC AAT 1200
 Gln Asn Gln Glu Pro Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Asn
 385 390 395 400
 TAA 1203
 (2) INFORMATION FOR SEQ ID NO: 2:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 400 amino acids
 (B) TYPE: amino acid
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2
 Met Lys Lys Ile Thr Thr Ile Phe Val Val Leu Leu Met Thr Val Ala
 1 5 10 15
 Leu Phe Ser Ile Gly Asn Thr Thr Ala Ala Asp Asn Asp Ser Val Val
 20 25 30
 Glu Glu His Gly Gln Leu Ser Ile Ser Asn Gly Glu Leu Val Asn Glu
 35 40 45
 Arg Gly Glu Gln Val Gln Leu Lys Gly Met Ser Ser His Gly Leu Gln
 50 55 60
 Trp Tyr Gly Gln Phe Val Asn Tyr Glu Ser Met Lys Trp Leu Arg Asp
 65 70 75 80
 Asp Trp Gly Ile Asn Val Phe Arg Ala Ala Met Tyr Thr Ser Ser Gly
 85 90 95
 Gly Tyr Ile Asp Asp Pro Ser Val Lys Glu Lys Val Lys Glu Ala Val
 100 105 110
 Glu Ala Ala Ile Asp Leu Asp Ile Tyr Val Ile Ile Asp Trp His Ile
 115 120 125
 Leu Ser Asp Asn Asp Pro Asn Ile Tyr Lys Glu Glu Ala Lys Asp Phe
 130 135 140
 Phe Asp Glu Met Ser Glu Leu Tyr Gly Asp Tyr Pro Asn Val Ile Tyr
 145 150 155 160
 Glu Ile Ala Asn Glu Pro Asn Gly Ser Asp Val Thr Trp Gly Asn Gln
 165 170 175
 Ile Lys Pro Tyr Ala Glu Glu Val Ile Pro Ile Ile Arg Asn Asn Asp
 180 185 190
 Pro Asn Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser Gln Asp Val
 195 200 205
 His His Ala Ala Asp Asn Gln Leu Ala Asp Pro Asn Val Met Tyr Ala
 210 215 220
 Phe His Phe Tyr Ala Gly Thr His Gly Gln Asn Leu Arg Asp Gln Val
 225 230 235 240
 Asp Tyr Ala Leu Asp Gln Gly Ala Ala Ile Phe Val Ser Glu Trp Gly
 245 250 255
 Thr Ser Ala Ala Thr Gly Asp Gly Gly Val Phe Leu Asp Glu Ala Gln
 260 265 270
 Val Trp Ile Asp Phe Met Asp Glu Arg Asn Leu Ser Trp Ala Asn Trp
 275 280 285
 Ser Leu Thr His Lys Asp Glu Ser Ser Ala Ala Leu Met Pro Gly Ala
 290 295 300
 Asn Pro Thr Gly Gly Trp Thr Glu Ala Glu Leu Ser Pro Ser Gly Thr
 305 310 315 320
 Phe Val Arg Glu Lys Ile Arg Glu Ser Ala Ser Ile Pro Pro Ser Asp
 325 330 335
 Pro Thr Pro Pro Ser Asp Pro Gly Glu Pro Asp Pro Thr Pro Pro Ser
 340 345 350
 Asp Pro Gly Glu Tyr Pro Ala Trp Asp Pro Asn Gln Ile Tyr Thr Asn
 355 360 365
 Glu Ile Val Tyr His Asn Gly Gln Leu Trp Gln Ala Lys Trp Trp Thr
 370 375 380
 Gln Asn Gln Glu Pro Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Asn
 385 390 395 400
 (2) INFORMATION FOR SEQ ID NO: 3:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 49 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3
 TCACAGATCC TCGCGAATTG GTGCGGCCGC GTNGTNGARG ARCAYGGNC 49
 (2) INFORMATION FOR SEQ ID NO: 4:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 7 amino acids
 (B) TYPE: amino acid
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4
 Val Val Glu Glu His Gly Gln
 5
 (2) INFORMATION FOR SEQ ID NO: 5:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 20 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5
 CAGAGCAAGAG ATTACGCGC 20
 (2) INFORMATION FOR SEQ ID NO: 6:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6
 GTTTTCCCAG TCACGAC 17
 (2) INFORMATION FOR SEQ ID NO: 7:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 22 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7
 GCGGATAACA ATTTCACACA GG 22
 (2) INFORMATION FOR SEQ ID NO: 8:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 20 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8
 TGACCCGTAC GGTCCGTGGG 20
 (2) INFORMATION FOR SEQ ID NO: 9:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 22 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9
 GGCTCTTGAT TTTGTGTCCA CC 22
 (2) INFORMATION FOR SEQ ID NO: 10:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 71 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10
 GTAGGCTCAG TCATATGTTA CACATTGAAA GGGGAGGAGA ATCATGAAAA AGATAACTAC 60
 TATTTTTGTC G 71
 (2) INFORMATION FOR SEQ ID NO: 11:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 51 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11
 GTACCTCGCG GGTACCAAGC GGCCGCTTAA TTGAGTGGTT CCCACGGACC G 51
 (2) INFORMATION FOR SEQ ID NO: 12:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 1389 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: Bacillus agaradherens
 (B) STRAIN: AC13
 (ix) FEATURE:
 (A) NAME/KEY: CDS
 (B) LOCATION: 1..1389
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12
 ATG AAA AAG ATA ACT ACT ATT TTT GTC GTA TTG CTT ATG ACA GTG GCG 48
 Met Lys Lys Ile Thr Thr Ile Phe Val Val Leu Leu Met Thr Val Ala
 1 5 10 15
 TTG TTC AGT ATA GGA AAC ACG ACT GCT GCT GAT AAT GAT TCA GTT GTA 96
 Leu Phe Ser Ile Gly Asn Thr Thr Ala Ala Asp Asn Asp Ser Val Val
 20 25 30
 GAA GAA CAT GGG CAA TTA AGT ATT AGT AAC GGT GAA TTA GTC AAT GAA 144
 Glu Glu His Gly Gln Leu Ser Ile Ser Asn Gly Glu Leu Val Asn Glu
 35 40 45
 CGA GGC GAA CAA GTT CAG TTA AAA GGG ATG AGT TCC CAT GGT TTG CAA 192
 Arg Gly Glu Gln Val Gln Leu Lys Gly Met Ser Ser His Gly Leu Gln
 50 55 60
 TGG TAC GGT CAA TTT GTA AAC TAT GAA AGT ATG AAA TGG CTA AGA GAT 240
 Trp Tyr Gly Gln Phe Val Asn Tyr Glu Ser Met Lys Trp Leu Arg Asp
 65 70 75 80
 GAT TGG GGA ATA AAT GTA TTC CGA GCA GCA ATG TAT ACC TCT TCA GGA 288
 Asp Trp Gly Ile Asn Val Phe Arg Ala Ala Met Tyr Thr Ser Ser Gly
 85 90 95
 GGA TAT ATT GAT GAT CCA TCA GTA AAG GAA AAA GTA AAA GAG GCT GTT 336
 Gly Tyr Ile Asp Asp Pro Ser Val Lys Glu Lys Val Lys Glu Ala Val
 100 105 110
 GAA GCT GCG ATA GAC CTT GAT ATA TAT GTG ATC ATT GAT TGG CAT ATC 384
 Glu Ala Ala Ile Asp Leu Asp Ile Tyr Val Ile Ile Asp Trp His Ile
 115 120 125
 CTT TCA GAC AAT GAC CCA AAT ATA TAT AAA GAA GAA GCG AAG GAT TTC 432
 Leu Ser Asp Asn Asp Pro Asn Ile Tyr Lys Glu Glu Ala Lys Asp Phe
 130 135 140
 TTT GAT GAA ATG TCA GAG TTG TAT GGA GAC TAT CCG AAT GTG ATA TAC 480
 Phe Asp Glu Met Ser Glu Leu Tyr Gly Asp Tyr Pro Asn Val Ile Tyr
 145 150 155 160
 GAA ATT GCA AAT GAA CCG AAT GGT AGT GAT GTT ACG TGG GGC AAT CAA 528
 Glu Ile Ala Asn Glu Pro Asn Gly Ser Asp Val Thr Trp Gly Asn Gln
 165 170 175
 ATA AAA CCG TAT GCA GAG GAA GTC ATT CCG ATT ATT CGT AAC AAT GAC 576
 Ile Lys Pro Tyr Ala Glu Glu Val Ile Pro Ile Ile Arg Asn Asn Asp
 180 185 190
 CCT AAT AAC ATT ATT ATT GTA GGT ACA GGT ACA TGG AGT CAG GAT GTC 624
 Pro Asn Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser Gln Asp Val
 195 200 205
 CAT CAT GCA GCT GAT AAT CAG CTT GCA GAT CCT AAC GTC ATG TAT GCA 672
 His His Ala Ala Asp Asn Gln Leu Ala Asp Pro Asn Val Met Tyr Ala
 210 215 220
 TTT CAT TTT TAT GCA GGG ACA CAT GGT CAA AAT TTA CGA GAC CAA GTA 720
 Phe His Phe Tyr Ala Gly Thr His Gly Gln Asn Leu Arg Asp Gln Val
 225 230 235 240
 GAT TAT GCA TTA GAT CAA GGA GCA GCG ATA TTT GTT AGT GAA TGG GGA 768
 Asp Tyr Ala Leu Asp Gln Gly Ala Ala Ile Phe Val Ser Glu Trp Gly
 245 250 255
 ACA AGT GCA GCT ACA GGT GAT GGT GGC GTG TTT TTA GAT GAA GCA CAA 816
 Thr Ser Ala Ala Thr Gly Asp Gly Gly Val Phe Leu Asp Glu Ala Gln
 260 265 270
 GTG TGG ATT GAC TTT ATG GAT GAA AGA AAT TTA AGC TGG GCC AAC TGG 864
 Val Trp Ile Asp Phe Met Asp Glu Arg Asn Leu Ser Trp Ala Asn Trp
 275 280 285
 TCT CTA ACG CAT AAA GAT GAG TCA TCT GCA GCG TTA ATG CCA GGT GCA 912
 Ser Leu Thr His Lys Asp Glu Ser Ser Ala Ala Leu Met Pro Gly Ala
 290 295 300
 AAT CCA ACT GGT GGT TGG ACA GAG GCT GAA CTA TCT CCA TCT GGT ACA 960
 Asn Pro Thr Gly Gly Trp Thr Glu Ala Glu Leu Ser Pro Ser Gly Thr
 305 310 315 320
 TTT GTG AGG GAA AAA ATA AGA GAA TCA GCA TCT ATT CCG CCA AGC GAT 1008
 Phe Val Arg Glu Lys Ile Arg Glu Ser Ala Ser Ile Pro Pro Ser Asp
 325 330 335
 CCA ACA CCG CCA TCT GAT CCA GGA GAA CCG GAT CCA ACG CCC CCA AGT 1056
 Pro Thr Pro Pro Ser Asp Pro Gly Glu Pro Asp Pro Thr Pro Pro Ser
 340 345 350
 GAT CCA GGA AAG TAT CCA GCA TGG GAT CCA AAT CAA ATT TAC ACA AAT 1104
 Asp Pro Gly Lys Tyr Pro Ala Trp Asp Pro Asn Gln Ile Tyr Thr Asn
 355 360 365
 GAA ATT GTG TAC CAT AAC GGC CAG CTA TGG CAA GCA AAA TGG TGG ACA 1152
 Glu Ile Val Tyr His Asn Gly Gln Leu Trp Gln Ala Lys Trp Trp Thr
 370 375 380
 CAA AAT CAA GAG CCA GGT GAC CCG TAC GGT CCG TGG GAA CCA CTC AAA 1200
 Gln Asn Gln Glu Pro Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Lys
 385 390 395 400
 TCT GAT CCA GAT TCA GGA GAA CCG GAT CCA ACG CCC CCA AGT GAT CCA 1248
 Ser Asp Pro Asp Ser Gly Glu Pro Asp Pro Thr Pro Pro Ser Asp Pro
 405 410 415
 GGA GAA TAT CCA GCA TGG GAC CCA ACG CAA ATT TAC ACA GAT GAA ATT 1296
 Gly Glu Tyr Pro Ala Trp Asp Pro Thr Gln Ile Tyr Thr Asp Glu Ile
 420 425 430
 GTG TAC CAT AAC GGC CAG CTA TGG CAA GCC AAA TGG TGG ACA CAA AAT 1344
 Val Tyr His Asn Gly Gln Leu Trp Gln Ala Lys Trp Trp Thr Gln Asn
 435 440 445
 CAA GAG CCA GGT GAC CCA TAC GGT CCG TGG GAA CCA CTC AAT TAA 1389
 Gln Glu Pro Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Asn *
 450 455 460
 (2) INFORMATION FOR SEQ ID NO: 13:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 462 amino acids
 (B) TYPE: amino acid
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13
 Met Lys Lys Ile Thr Thr Ile Phe Val Val Leu Leu Met Thr Val Ala
 1 5 10 15
 Leu Phe Ser Ile Gly Asn Thr Thr Ala Ala Asp Asn Asp Ser Val Val
 20 25 30
 Glu Glu His Gly Gln Leu Ser Ile Ser Asn Gly Glu Leu Val Asn Glu
 35 40 45
 Arg Gly Glu Gln Val Gln Leu Lys Gly Met Ser Ser His Gly Leu Gln
 50 55 60
 Trp Tyr Gly Gln Phe Val Asn Tyr Glu Ser Met Lys Trp Leu Arg Asp
 65 70 75 80
 Asp Trp Gly Ile Asn Val Phe Arg Ala Ala Met Tyr Thr Ser Ser Gly
 85 90 95
 Gly Tyr Ile Asp Asp Pro Ser Val Lys Glu Lys Val Lys Glu Ala Val
 100 105 110
 Glu Ala Ala Ile Asp Leu Asp Ile Tyr Val Ile Ile Asp Trp His Ile
 115 120 125
 Leu Ser Asp Asn Asp Pro Asn Ile Tyr Lys Glu Glu Ala Lys Asp Phe
 130 135 140
 Phe Asp Glu Met Ser Glu Leu Tyr Gly Asp Tyr Pro Asn Val Ile Tyr
 145 150 155 160
 Glu Ile Ala Asn Glu Pro Asn Gly Ser Asp Val Thr Trp Gly Asn Gln
 165 170 175
 Ile Lys Pro Tyr Ala Glu Glu Val Ile Pro Ile Ile Arg Asn Asn Asp
 180 185 190
 Pro Asn Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser Gln Asp Val
 195 200 205
 His His Ala Ala Asp Asn Gln Leu Ala Asp Pro Asn Val Met Tyr Ala
 210 215 220
 Phe His Phe Tyr Ala Gly Thr His Gly Gln Asn Leu Arg Asp Gln Val
 225 230 235 240
 Asp Tyr Ala Leu Asp Gln Gly Ala Ala Ile Phe Val Ser Glu Trp Gly
 245 250 255
 Thr Ser Ala Ala Thr Gly Asp Gly Gly Val Phe Leu Asp Glu Ala Gln
 260 265 270
 Val Trp Ile Asp Phe Met Asp Glu Arg Asn Leu Ser Trp Ala Asn Trp
 275 280 285
 Ser Leu Thr His Lys Asp Glu Ser Ser Ala Ala Leu Met Pro Gly Ala
 290 295 300
 Asn Pro Thr Gly Gly Trp Thr Glu Ala Glu Leu Ser Pro Ser Gly Thr
 305 310 315 320
 Phe Val Arg Glu Lys Ile Arg Glu Ser Ala Ser Ile Pro Pro Ser Asp
 325 330 335
 Pro Thr Pro Pro Ser Asp Pro Gly Glu Pro Asp Pro Thr Pro Pro Ser
 340 345 350
 Asp Pro Gly Lys Tyr Pro Ala Trp Asp Pro Asn Gln Ile Tyr Thr Asn
 355 360 365
 Glu Ile Val Tyr His Asn Gly Gln Leu Trp Gln Ala Lys Trp Trp Thr
 370 375 380
 Gln Asn Gln Glu Pro Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Lys
 385 390 395 400
 Ser Asp Pro Asp Ser Gly Glu Pro Asp Pro Thr Pro Pro Ser Asp Pro
 405 410 415
 Gly Glu Tyr Pro Ala Trp Asp Pro Thr Gln Ile Tyr Thr Asp Glu Ile
 420 425 430
 Val Tyr His Asn Gly Gln Leu Trp Gln Ala Lys Trp Trp Thr Gln Asn
 435 440 445
 Gln Glu Pro Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Asn
 450 455 460
 (2) INFORMATION FOR SEQ ID NO: 14:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 46 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
 CCTCGCGAGG TACCAGCGGC CGCGTACCAC CAATTAAGTA TGGTAC 46