# Patent

## Title
Polypeptides of botryosphaeria rhodina

## Abstract

The invention relates to functional polypeptides secreted from Botryospaeria rhodina CBS 274.96.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/573,261 filed on Mar. 13, 2007 (now U.S. Pat. No. 7,666,649) which claims priority of 35 U.S.C. 371 national application of PCT/DK2005/00519 filed Aug. 6, 2005, which claims priority or the benefit under 35 U.S.C. 119 of Danish application nos. PA 2004 01197 and PA 2004 01215 filed Aug. 6, 2004 and Aug. 11, 2004 and U.S. provisional application No. 60/622,984 filed Oct. 28, 2004, the contents of which are fully incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to functional polypeptides encoded by polynucleotides comprised in the mRNA of Diplodia gossypina, syn. Botryospaeria rhodina deposited under deposit accession number CBS 274.96. The invention relates further to the polynucleotides and constructs of such polynucleotides encoding such polypeptides or facilitating their expression as well as to method for preparing the polypeptide. Still further the invention relates to compositions comprising the polypeptide and to uses of the polypeptide.
BACKGROUND OF THE INVENTION
Botryosphaeria rhodina (Berk. & M. A. Curtis) Arx, (syn. Diplodia gossypina Cooke). Very few reports have been published concerning the characterization of this organism; Selbmann et al., 2003 report exopolysaccharide production from Botryosphaeria rhodina (Selbmann L, Stingele F, and Petruccioli M (2003) Exopolysaccharide production by filamentous fungi: the example of Botryosphaeria rhodina. Antonie van Leeuwenhoek, 84:2, pp. 135-145. A single patent exists on the use of Botryosphaeria rhodina to hydroxylate beta Damascone as a flavouring agent for tobacco (CH654567-A). Jasmonic acid can also be produced by fermenting the organism (EP1118672-A). The presence of cellobiohydrolases (WO 03/000941-A2) and lactase (WO 01/49878-A) have been previously reported.
In the pursuit of novel enzymes it is also known to screen for such new enzymes by subjecting potential candidates to specific enzyme assays. This approach is limited to the availability of enzyme assays and does not allow the identification of functional enzymes or polypeptides for which the activity is still unknown.
Further, whole genome sequencing is a known method to obtain the information on all genes from a given microorganism e.g. as described in Fleischmann et al.; Whole genome sequences and assembly of Haemophilus influenzae Rd; Nature 269: 496-512; (1995).
Most enzymes for industrial use are enzymes which are secreted to the medium by a microorganism. However, only a few percent of a microorganisms' genome encodes secreted proteins. For example only approx. 4% of the Bacillus subtilis genome or its closest relatives encode secreted proteins (Van Dijl et al.: Protein transport pathways in Bacillus subtilis: a genome-based road map; in “Basillus subtilis and it's closest relatives”—From genes to cells; p. 337-355; A. L. Sonenshein (ed.); ASM Press 2002).
One disadvantage of genome sequencing is that the vast majority of the obtained sequences encode non secreted proteins.
An additional disadvantage of gemomic sequencing particular to eukaryotes such as fungi is that the genome size is many times larger than a bacterial genome making gene discovery by this method more costly and time consuming.
The random sequencing of cDNAs (Expressed sequence tags or ESTs) is another approach that allows for discovery of secreted proteins. In general, EST approaches suffer two drawbacks with regard to secreted protein identification; 1) Depending on the induction conditions used for the cDNA library sequenced, very few, typically between 0.5%-15% or even 1 and 5% of the cDNAs encode secreted proteins. 2) The clones all come from a cDNA pool derived from mRNAs that are present in the organism in proportion to the induction level of each particular gene.
Also known is signal trapping which is a method to identify genes including nucleotides encoding a signal peptide using a translational fusion to an extracellular reporter gene lacking its own signal (WO 01/77315).
SUMMARY OF THE INVENTION
The genome of a microorganism contains thousands of different genes; some encoding polypeptides some coding for RNAs. Only a limited number of the genes in the genome of a microorganism encode functional polypeptides which are secreted by the microorganism to the surrounding medium serving an external purpose for the microorganism.
Such polypeptides are interesting for industry from the point of view that such polypeptides may be produced in considerable amounts in continuous processes without destroying the cells producing the polypeptides.
It is an object of the present invention to identify and provide polypeptides secreted from Botryosphaeria rhodina deposited under deposit accession number CBS 274.96 which have functional purpose for the Botryosphaeria rhodina because such polypeptides may not only be used for industrial purposes but they may also be produced in industrially relevant processes and amounts.
The present invention provides in a first aspect an isolated polypeptide selected from the group consisting of:

    (a) a polypeptide comprising an amino acid sequence which has at least 90% identity with a sequence of a mature polypeptide comprised in the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 32, 34, 36, 38, 40 and 42; and
    (b) a polypeptide which is encoded by a nucleotide sequence which hybridize under high stringency conditions with a polynucleotide probe selected from the group consisting of
    
        (i) the complementary strand to a nucleotide sequence selected from the group of regions of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41 encoding a mature polypeptide.
        (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from the group of regions of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41 encoding a mature polypeptide

wherein the polypeptide has a function of the corresponding mature polypeptides comprised in the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 32, 34, 36, 38, 40 and 42.

    
    


In a further aspect the invention provides an isolated enzyme selected from the group consisting of:

    (a) an enzyme comprising an amino acid sequence which has at least 90% identity with the amino acid sequence of a mature enzyme selected from the group consisting of xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase secreted from the strain of Botryosphaeria rhodina deposited under CBS accession No. 274.96
    (b) a polypeptide which is encoded by a nucleotide sequence which hybridize under high stringency conditions with a polynucleotide probe selected from the group consisting of
    
        (i) the complementary strand to a nucleotide sequence comprised in the strain of Botryosphaeria rhodina Deposited under CBS accession No. 274.96 encoding a mature enzyme selected from the group consisting of xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61 D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase secreted from that strain;
        ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences comprised in the strain of Botryosphaeria rhodina Deposited under CBS accession No. 274.96 encoding a mature enzyme selected from the group consisting of xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase secreted from that strain;

wherein the enzyme have a function selected from xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase.

    
    


In further aspects the invention provides a polynucleotide encoding the polypeptide of the invention; a nucleotide construct comprising the polynucleotide encoding the polypeptide, operably linked to one or more control sequences that direct the production of the polypeptide in a host cell; a recombinant expression vector comprising the nucleotide construct of the invention and to a recombinant host cell comprising the nucleotide construct of the invention.
In still further aspects the invention provides a method of preparing a polypeptide of the invention comprising:

    (a) cultivating a strain comprising a nucleotide sequence encoding a polypeptide of the invention which strain is capable of expressing and secreting the polypeptide and
    (b) recovering the polypeptide.


In still further aspects the invention provide a composition comprising a polypeptide of the invention and a method for preparing such a composition comprising admixing the polypeptide of the invention with an excipient.
In still further aspects the invention provides use of the polypeptide of the invention or a composition comprising said polypeptide in various applications.
Sequence Listing
The present application contains information in the form of a sequence listing, which is appended to the application and also submitted on a data carrier accompanying this application. The contents of the data carrier are fully incorporated herein by reference. The regions of sequences selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41 which encode a mature polypeptide, encodes the mature polypeptides of sequences selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 32, 34, 36, 38, 40 and 42 respectively. The region of SEQ ID NO: 1 encoding a mature polypeptide thus encodes the mature polypeptide sequence comprised in SEQ ID NO:2, the region of SEQ ID NO:3 encoding a mature polypeptide encode the mature polypeptide comprised in SEQ ID NO:4 and so on.



BRIEF DESCRIPTION OF DRAWINGS
No drawings.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “group ADNA” as used hereinafter means a group of nucleotide sequences consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41. Hence when referral is made to a nucleotide sequence comprised in or selected from group of sequences consisting of “group ADNA “(or just comprised in or selected from “group ADNA”), it means that the sequence is comprised in or selected from the group of nucleotide sequences consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41.
The term “group EDNA” as used hereinafter means a group of nucleotide sequences consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 33, 35, 37, 39 and 41. Hence when referral is made to a nucleotide sequence comprised in or selected from group of sequences consisting of “group EDNA “(or just comprised in or selected from “group EDNA”), it means that the sequence is comprised in or selected from the group of nucleotide sequences consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 33, 35, 37, 39 and 41.
Like wise the term “group Bpolypeptide” as used hereinafter means a group of polypeptide sequences consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 32, 34, 36, 38, 40 and 42. Hence when referral is made to a polypetide sequence comprised in or selected from the group of sequences consisting of “group BPolypeptide” (or just comprised in or selected from “group BPolypeptide”) it means that the sequence is comprised in or selected from the group of polypeptide sequences consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 32, 34, 36, 38, 40 and 42.
Like wise the term “group Dpolypeptide” as used hereinafter means a group of polypeptide sequences consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 34, 36, 38, 40 and 42. Hence when referral is made to a polypetide sequence comprised in or selected from the group of sequences consisting of “group Dpolypeptide” (or just comprised in or selected from “group Dpolypeptide”) it means that the sequence is comprised in or selected from the group of polypeptide sequences consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 34, 36, 38, 40 and 42.
The term “identity” as used herein, is to be understood as the homology between two amino acid sequences or between two nucleotide sequences. For purposes of the present invention, the degree of identity between two amino acid sequences is determined by using AlignX in the program of Vector NTI ver. 7.1 (Informax inc., 7600 Wisconsin Avenue, Suite #1100, Bethesda, Md. 20814, USA). Amino acid alignment is created using the Clustal W algorithm (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). The following additional parameters are used: Gap opening penalty of 10, Gap extension penalty of 0.05, Gap separation penalty range of 8. Pairwise alignment parameters were Ktuple=1, gap penalty=3, gap length opening penalty=10, gap extension penalty=0.1, window size=5 and diagonals=5. The degree of identity between two nucleotide sequences is determined using the same algorithm and software package as described above for example with the following settings: Gap penalty of 10, and gap length penalty of 10. Pairwise alignment parameters is Ktuple=3, gap penalty=3 and windows=20.
The term “functional polypeptide” as used herein in the context of the present invention means a polypeptide which can be expressed and secreted by a cell and which constitutes an operational unit capable of operating in accordance with the function it is designed to fulfill by the cell. Optionally, co-factors may be required for the polypeptide to adopt the intended function. One example of functional polypeptides is catalytically active polypeptides or enzymes which help the cell catalyzing reactions in the environment surrounding the cell. Another example could be polypeptides which serve as signal substance. Further examples are polypeptides which function as sensors (receptors) for environmental parameters (chemicals in the environment surrounding the cell) or polypeptides, which are active against other organisms (antimicrobial (poly)peptides) or polypeptides, which contributes to the structural integrity of the cell.
The term “mature region” as used herein about portion of an amino acid sequences or polypeptide means the portion or region or domain or section of the amino acid sequences or polypeptide which is the mature functional polypeptide.
The term “region of nucleotide sequence encoding a mature polypeptide” as used herein means the region of a nucleotide sequence counting from the triplet encoding the first amino acid of a mature polypeptide to the last triplet encoding the last amino acid of a mature polypeptide.
The term “GH” as used about certain enzymes of the present invention, as for example “GH10”, is a family classification system for glycosyl hydrolase enzymes made by B. Henrissat. The number following the GH each denotes distinct families. This classification system is well known to the skilled person. See Henrissat B., A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280:309-316 (1991); Henrissat B., Bairoch A, New families in the classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 293:781-788 (1993); Henrissat B., Bairoch A., Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696 (1996); Davies G., Henrissat B., Structures and mechanisms of glycosyl hydrolases, Structure 3:853-859 (1995).
Polypeptides of the Invention
The polypeptides of the invention are all polypeptides secreted by Botryosphaeria rhodina CBS 274.96 with the purpose of serving a function for that particular cell.
Among the thousands of potential genes in the genome of Botryosphaeria rhodina CBS 274.96 the polynucleotides of this genome encoded 21 secreted functional mature polypeptides comprised in group BPolypeptide, which were determined to be functional, that is translated into functional polypeptides and secreted by the chosen host cell.
Accordingly, Botryosphaeria rhodina CBS 274.96 expresses and secretes the functional mature polypeptides comprised in group BPolypeptide, and in the genome of that particular strain, the regions of sequences of group ADNA, encoding a mature polypeptide are the genes encoding the mature polypeptides comprised in the sequences of group BPolypeptide. Further in a particular embodiment the genes encoding the mature polypeptides comprised in the sequences of of group BPolypeptide can all be expressed and their corresponding mature polypeptides can be secreted when culturing an E. coli host transformed with polynucleotides comprising those regions of the sequences of group ADNA encoding a mature polypeptide. By comparing homology or identity of the sequences of the 21 polypeptide sequences to known sequences the particular function of the polypeptides were annotated. At least 14 of the 21 secreted functional polypeptides were determined to be enzymes and/or enzyme like.
The invention thus provides an isolated polypeptide selected from the group consisting of:

    (a) a polypeptide having an amino acid sequence which has at least 90% identity with an amino acid sequence selected from the group consisting of the mature polypeptides comprised in group BPolypeptide and
    (b) a polypeptide which is encoded by a nucleotide sequence which hybridize under high stringency conditions with a polynucleotide probe selected from the group consisting of
    
        (i) the complementary strand to a nucleotide sequence selected from the group consisting of the regions of group ADNA sequences encoding a mature polypeptide,
        (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from the group consisting of the regions of group ADNA sequences encoding a mature polypeptide;

wherein the polypeptide exhibits the function of the corresponding mature polypeptides of group BPolypeptide.

    
    


In one particular embodiment the polypeptide of the invention is selected among the enzymes secreted by Botryosphaeria rhodina deposited under CBS accession No. 274.96 and isolated by the present inventors, i.e. the group of enzymes consisting of xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase.
The invention also provides an isolated enzyme selected from the group consisting of:

    (a) an enzyme comprising an amino acid sequence which has at least 90% identity with the amino acid sequence of a mature enzyme selected from the group consisting of xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase secreted from the strain of Botryosphaeria rhodina Deposited under CBS accession No. 274.96 and
    (b) a polypeptide which is encoded by a nucleotide sequence which hybridize under high stringency conditions with a polynucleotide probe selected from the group consisting of
    
        (i) the complementary strand to a nucleotide sequence comprised in the strain of Botryosphaeria rhodina Deposited under CBS accession No. 274.96 encoding a mature enzyme selected from the group consisting of xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61 D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase secreted from that strain;
        ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences comprised in the strain of Botryosphaeria rhodina Deposited under CBS accession No. 274.96 encoding a mature enzyme selected from the group consisting of xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase secreted from that strain and

wherein the enzyme have a function selected from xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase.

    
    


In a particular embodiment the enzyme is an isolated enzyme selected from the group consisting of:

    (a) an enzyme having an amino acid sequence which has at least 90% identity with an amino acid sequence selected from mature enzymes comprised in the sequences of group DPolypeptide and
    (b) an enzyme which is encoded by a nucleotide sequence which hybridize under high stringency conditions with a polynucleotide probe selected from the group consisting of
    
        (i) the complementary strand to a nucleotide sequence selected from the group consisting of the regions of group EDNA sequences encoding a mature enzyme,
        (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from the group consisting of the regions of group EDNA sequences encoding a mature enzyme;

wherein the enzyme exhibits the function of the corresponding mature enzyme of group DPolypeptide.

    
    


The polypeptide of the invention is an isolated polypeptide, preferably the preparation of the polypeptide of the invention contains at the most 90% by weight of other polypeptide material with which it may be natively associated (lower percentages of other polypeptide material are preferred, e.g. at the most 80% by weight, at the most 60% by weight, at the most 50% by weight, at the most 40% at the most 30% by weight, at the most 20% by weight, at the most 10% by weight, at the most 9% by weight ,at the most 8% by weight, at the most 6% by weight, at the most 5% by weight, at the most 4% at the most 3% by weight, at the most 2% by weight, at the most 1% by weight and at the most 1⁄2% by weight). Thus, it is preferred that the isolated polypeptide of the invention is at least 92% pure, i.e. that the polypeptide of the invention constitutes at least 92% by weight of the total polypeptide material present in the preparation, and higher percentages are preferred such as at least 94% pure, at least 95% pure, at least 96% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, and at the most 99.5% pure. In particular, it is preferred that the polypeptide of the invention is in “essentially pure form”, i.e. that the polypeptide preparation is essentially free of other polypeptide material with which it is natively associated. This can be accomplished, for example, by preparing the polypeptide of the invention by means of well-known recombinant methods.
The polypeptide of the invention of the invention may be synthetically made, naturally occurring or a combination thereof. In a particular embodiment the polypeptide of the invention may be obtained from a microorganism such as a prokaryotic cell, an archaeal cell or a eukaryotic cell. The cell may further have been modified by genetic engineering
In a particular embodiment, the polypeptide of the invention is an enzyme exhibiting optimum enzyme activity at a temperature within the range from about 10° C. to about 80° C., particularly in the range from about 20° C. to about 60° C.
In a particular embodiment the polypeptide of the invention is an enzyme, which is functionally stabile at a temperature of up to 100° C., in particular up to 80° C., more particularly up to 60° C.
In a particular embodiment the polypeptide of the invention is an enzyme exhibiting at least 20%, in particular at least 40%, such as at least 50%, in particular at least 60%, such as at least 70%, more particularly at least 80%, such as at least 90%, most particularly at least 95%, such as about or at least 100% of the enzyme activity of an enzyme selected from mature enzymes comprised in group BPolypeptide.
In a particular embodiment the polypeptide of the invention comprises, contains or consists of an amino acid sequence which has at least 90% identity with a polypeptide sequence selected from the group consisting of mature polypeptides comprised in group
BPolypeptide; particularly at least 95%, e.g. at least 96%, such as at least 97%, and even more particularly at least 98%, such as at least 99% or even 100% identity.
In another particular embodiment the polypeptide of the invention comprises, contains or consists of an amino acid sequence, which has at least 50% identity with a polypeptide sequence selected from the group consisting of mature polypeptides comprised in group
BPolypeptide; particularly at least 60%, particularly at least 65%, particularly at least 70%, particularly at least 75%, particularly at least 80%, and even more particularly at least 85% identity.
In a particular embodiment, the amino acid sequence of the polypeptide of the invention differs by at the most ten amino acids (e.g. by ten amino acids), in particular by at the most five amino acids (e.g. by five amino acids), such as by at the most four amino acids (e.g. by four amino acids), e.g. by at the most three amino acids (e.g. by three amino acids), in particular by at the most two amino acids (e.g. by two amino acids), such as by one amino acid from the mature polypeptides comprised in group BPolypeptide.
The polypeptide of the invention may be a wild-type polypeptide isolated from a natural source such as the strain Botryosphaeria rhodina CBS 274.96 or another wild type strain, however the present invention also encompass artificial variants, where a polypeptide of the invention has been mutated for example by adding, substituting and/or deleting one or more amino acids from said polypeptide while retaining the function of the polypeptide and/or other properties.
Hence, the polypeptide of the invention may be an artificial variant, wherein at least one substitution, deletion and/or insertion of an amino acid has been made to an amino acid sequence comprising or consisting of the mature polypeptide comprised in group BPolypeptide.
The polypeptides of the invention also include functional fragments of the amino acid sequences described herein and nucleic acids encoding functional fragments of the amino acid sequences described herein, including fragments of the mature enzymes secreted from the strain of Botryosphaeria rhodina Deposited under CBS accession No. 274.96, as described herein, including fragment of an enzyme selected from the group consisting of xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase secreted from the strain of Botryosphaeria rhodina Deposited under CBS accession No. 274.96.
Artificial variants may be constructed by standard techniques known in the art usually followed by screening and/or characterization. Standard techniques includes classical mutagenesis, e.g. by UV irradiation of the cells or treatment of cells with chemical mutagens as described by Gerhardt et al. (1994); in vivo gene shuffling as described in WO 97/07205; in vitro shuffling as described by Stemmer, (1994) or WO 95/17413, random mutagenesis as described by Eisenstadt E. et al., (1994); PCR techniques as described by Poulsen et al. (1991); family shuffling as described by J. E. Ness, et al, Nature Biotechnology, vol. 17, pp. 893-896 (1999); site-directed mutagenesis as described by Sambrook et al. (1989), Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y. A general description of nucleotide substitution can be found in e.g. Ford et al., 1991, Protein Expression and Purification 2, p. 95-107.
Such standard genetic engineering methods may also be used prepare a diversified library of variant nucleotide sequences from the genes encoding one or more parent enzymes of the invention, expressing the enzyme variants in a suitable host cell and selecting a preferred variant(s). A diversified library can be established by a range of techniques known to the art (Reetz M T; Jaeger K E, in Biocatalysis—from Discovery to Application edited by Fessner W D, Vol. 200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p.389-391, 1994; Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000, 1997; or Yano et al., Proc. Natl. Acad. Sci., USA, vol. 95, pp 5511-5515, 1998).
In a particular embodiment of the invention, amino acid changes (in the artificial variant as well as in wild-type enzyme) are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/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 by changing net charge or another function, 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 (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions which do not generally alter and or impair the function of a protein are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these in reverse.
In a particular embodiment the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may be performed, which improve the thermal stability of the enzyme, which alter the substrate specificity, which changes the pH optimum, and the like.
Particularly, the number of such substitutions, deletions and/or insertions in the polypeptide of the invention, particularly in those polypeptides selected from the group consisting of mature polypeptides comprised in group BPolypeptide to produce an artificial variant is at the most 10, such as at the most 9, e.g. at the most 8, more preferably at the most 7, e.g. at the most 6, such as at the most 5, most preferably at the most 4, e.g. at the most 3, such as at the most 2, in particular at the most 1.
In a particular embodiment the artificial variant is a variant, which has an altered, preferably reduced, immunogenicity, especially allergenicity, in animals including man as compared to a parent enzyme. The term “immunogenicity” in this context is to be understood as the artificial variant capability of invoking a an altered, in particular reduced, immunological response when administered to an animal, including intravenous, cutaneous, subcutaneous, oral and intratracheal administration. The term “immunological response” in this context means that the administration of the artificial variant causes an alteration in the immunoglobulin levels in the animal body, such as in IgE, IgG and IgM or an alteration in the cytokine level in the animal body. Methods for mapping immunogenic/antigenic epitopes of a protein, preparing variants with altered immunogenicity and methods for measuring an immunological response is well known to the art and are described e.g. in WO 92/10755, WO 00/26230, WO 00/26354 and WO 01/31989. The term “allergenicity” in this context is to be understood as the artificial variant ability of invoking an altered, in particular reduced, production of IgE in an animal as well as the ability to bind IgE from said animal. Particularly allergenicity arising from intratracheal administration of the polypeptide variant to the animal is particularly of interest (also known as respiratory allergenicity).
In a further embodiment, the polypeptide of the invention is a polypeptide which is encoded by nucleotide sequences which hybridize under at least high stringency conditions, particularly under very high stringency conditions with a polynucleotide probe selected from the group consisting of

    (i) the complementary strand to a nucleotide sequence selected from the group consisting of the regions of group ADNA sequences encoding a mature polypeptide,
    (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from the group consisting of the regions of group ADNA sequences encoding a mature polypeptide;
    (iii) a fragment of (i) or (ii) encoding a secreted polypeptide having the function of the corresponding mature polypeptide comprised in group BPolypeptide.

(J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

In particular, the polypeptide of the invention is encoded by a polynucleotide comprising a nucleotide sequence selected from the group of regions of group ADNA sequences encoding a mature polypeptide or a sequences differing there from by virtue of the degeneracy of the genetic code. More particularly, the polypeptide of the invention is encoded by a polynucleotide consisting of a nucleotide sequence selected from the group of regions of group ADNA sequences encoding a mature polypeptide or a sequence differing there from by virtue of the degeneracy of the genetic code.



The nucleotide sequences of regions of group ADNA sequences encoding a mature polypeptide or a subsequence thereof, as well as the amino acid sequences of the mature polypeptides comprised in group BPolypeptide or a fragment thereof, may be used to design a polynucleotide probe to identify and clone DNA encoding enzymes of the invention from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, more preferably at least 35 nucleotides in length, such as at least 70 nucleotides in length. It is; however, preferred that the polynucleotide probe is at least 100 nucleotides in length. For example, the polynucleotide probe may be at least 200 nucleotides in length, at least 300 nucleotides in length, at least 400 nucleotides in length or at least 500 nucleotides in length. Even longer probes may be used, e.g., polynucleotide probes which are at least 600 nucleotides in length, at least 700 nucleotides in length, at least 800 nucleotides in length, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labelled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin).
Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA, which hybridizes with the probes described above and which encodes enzymes of the invention. 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 materials. In order to identify a clone or DNA which has the required homology and/or identity or is homologous and/or identical with nucleotides selected from regions of group ADNA sequences encoding a mature polypeptide, the carrier material with the immobilized DNA is used in a Southern blot.
For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labelled polynucleotide probe which again hybridizes to a nucleotide sequence selected from regions of group ADNA sequences encoding a mature polypeptide under high to very high stringency conditions. Molecules to which the polynucleotide probe hybridizes under these conditions may be detected using X-ray film or by any other method known in the art. Whenever the term “polynucleotide probe” is used in the present context, it is to be understood that such a probe contains at least 15 nucleotides.
In an interesting embodiment, the polynucleotide probe is the complementary strand of a nucleotide sequence selected from regions of group ADNA sequences encoding a mature polypeptide.
In another interesting embodiment, the polynucleotide probe is the complementary strand of a nucleotide sequence which encodes an enzyme selected from group BPolypeptide. In a further interesting embodiment, the polynucleotide probe is the complementary strand of a mature polypeptide coding region of a nucleotide sequence selected from regions of group ADNA sequences encoding a mature polypeptide.
For long probes of at least 100 nucleotides in length, high to very high stringency conditions are defined as pre-hybridization and hybridization at 42° C. in 5×SSPE, 1.0% SDS, 5× Denhardt's solution, 100 microgram/ml sheared and denatured salmon sperm DNA, following standard Southern blotting procedures. Preferably, the long probes of at least 100 nucleotides do not contain more than 1000 nucleotides. For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 0.1×SSC, 0.1% SDS at 60° C. (high stringency), in particular washed three times each for 15 minutes using 0.1×SSC, 0.1% SDS at 68° C. (very high stringency).
Although not particularly preferred, it is contemplated that shorter probes, e.g. probes which are from about 15 to 99 nucleotides in length, such as from about 15 to about 70 nucleotides in length, may be also be used. For such short probes, stringency conditions are defined as pre-hybridization, hybridization, and washing post-hybridization at 5° C. to 10° C. below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.
For short probes which are about 15 nucleotides to 99 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated Tm.
SEQ ID NO: 2 Xylanase GH10
In a particular embodiment the polypeptide of the invention is a GH10 xylanase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a GH10 xylanase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature GH10 xylanase comprised in SEQ ID NO: 2. More specifically the mature GH10 xylanase comprise or consists of the sequences from position 1 to 291 of SEQ ID NO: 2. In the present context a GH10 xylanase is defined as an enzyme belonging to the EC 3.2.1.8 enzyme activity grouping. This grouping endohydrolyses 1,4-β-D-xylosidic linkages in xylans. The glycoside hydrolase family 10 (GH10) also comprises enzymes with two other known activities; endo-1,3-beta-xylanase (EC: 3.2.1.32); cellobiohydrolase (EC: 3.2.1.91).
SEQ ID NO: 4 Xylanase GH11
In a particular embodiment the polypeptide of the invention is a GH11 xylanase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a xylanase GH11 obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature GH11 xylanase comprised in SEQ ID NO: 4. More specifically the mature GH11 xylanase comprise or consists of the sequences from position 1 to 202 of SEQ ID NO: 4. In the present context a GH11 xylanase is defined as an enzyme beloning to the EC 3.2.1.8 enzyme activity grouping. This grouping endohydrolyses 1,4-β-D-xylosidic linkages in xylans. Glycoside hydrolase family 11 (GH11) comprises enzymes with only one known activity; xylanase (EC: 3.2.1.8).
SEQ ID NO: 6 Serine Esterase
In a particular embodiment the polypeptide of the invention is a serine esterase, in particular a cutinase or a lipase or a carboxyesterase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a serine esterase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature serine esterase comprised in SEQ ID NO: 6. More specifically the mature serine esterase comprise or consists of the sequences from position 1 to 352 of SEQ ID NO: 6. In the present context a serine esterase is defined as an enzyme capable of hydrolysing soluble esters in solution (esters which are not in micelle form). More specifically the serin esters are enzymes acting as cutinase (EC 3.1.1.50) or lipase (EC.3.1.1.3) or carboxyesterase capable of hydrolysing wax-esters, cutin, tracyl fats, oils and/or fatty acid chains. In particular the serine esterases contain the classical Ser, His, Asp triad of serine hydrolase, such as tri-acyl lipase/cutinase.
SEQ ID NO: 8 Candida B Type Lipase
In a particular embodiment the polypeptide of the invention is a Candida B type lipase (EC 3.1.1.3) comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a Candida B type Lipase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature Candida B type Lipase comprised in SEQ ID NO: 8. More specifically the mature Candida B type Lipase comprise or consists of the sequences from position 1 to 431 of SEQ ID NO: 8. In the present context the Candida B type Lipase is defined as an enzyme capable of hydrolysing triglycerides to diacyl glycerides and fatty acid anions, in particular triacylglycerol to diacylglycerol and a fatty acid anion.
SEQ ID NO: 10 Peroxidase
In a particular embodiment the polypeptide of the invention is a peroxidase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a peroxidase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature peroxidase comprised in SEQ ID NO: 10. More specifically the mature peroxidase comprises or consists of the sequences from position 1 to 185 of SEQ ID NO: 10. In the present context a peroxidase is defined as an enzyme belonging to defined as a group of enzymes that catalyze oxidation-reduction reactions. As such, they are classified as oxidoreductases. They are given the official EC number 1.11.1. Peroxidases reduce H2O2 to water while oxidizing a variety of substrates. Thus, peroxidases are oxidoreductases which use H2O2 as electron acceptor for catalyzing different oxidative reactions.
SEQ ID NO: 12 GH61A Polypeptide
In a particular embodiment the polypeptide of the invention is a GH61A polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a GH61A polypeptide obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature GH61A polypeptide comprised in SEQ ID NO: 12. More specifically the mature GH61A polypeptide comprises or consists of the sequences from position 1 to 218 of SEQ ID NO: 12. In the present context a GH 61A polypeptide is defined as a secreted polypeptide or protein providing one or more of the group of effects selected from:

    1) Enhancing degradation of cellulosic materials when used in conjunction with a cellulase or a mixture of cellulases.
    2) Increasing solubility of enzymes.
    3) Increasing the stability of enzymes
    4) Reducing enzyme inhibition.

SEQ ID NO: 14 GH 61B Polypeptide



In a particular embodiment the polypeptide of the invention is a GH 61 B polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a GH 61B polypeptide obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature GH 61B polypeptide comprised in SEQ ID NO: 14. More specifically the mature GH 61B polypeptide comprises or consists of the sequences from position 1 to 249 of SEQ ID NO: 14. In the present context a GH 61B polypeptide is defined as a secreted polypeptide or protein providing one or more of the group of effects selected from:

    1) Enhancing degradation of cellulosic materials when used in conjunction with a cellulase or a mixture of cellulases.
    2) Increasing solubility of enzymes.
    3) Increasing the stability of enzymes

SEQ ID NO: 16 GH 61C Polypeptide



In a particular embodiment the polypeptide of the invention is a GH 61 C polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a GH 61C polypeptide obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature GH 61C polypeptide comprised in SEQ ID NO: 16. More specifically the mature GH 61C polypeptide comprises or consists of the sequences from position 1 to 255 of SEQ ID NO: 16. In the present context a GH 61C polypeptide is defined as a secreted polypeptide or protein providing one or more of the group of effects selected from:

    1) Enhancing degradation of cellulosic materials when used in conjunction with a cellulase or a mixture of cellulases.
    2) Increasing solubility of enzymes.
    3) Increasing the stability of enzymes

SEQ ID NO: 18 GH 61D Polypeptide



In a particular embodiment the polypeptide of the invention is a GH 61 D polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a GH 61D polypeptide obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature GH 61 D polypeptide comprised in SEQ ID NO: 18. More specifically the mature GH 61D polypeptide comprises or consists of the sequences from position 1 to 205 of SEQ ID NO: 18. In the present context a GH 61C polypeptide is defined as a secreted polypeptide or protein providing one or more of the group of effects selected from:

    1) Enhancing degradation of cellulosic materials when used in conjunction with a cellulase or a mixture of cellulases.
    2) Increasing solubility of enzymes.
    3) Increasing the stability of enzymes

SEQ ID NO:20 Functional Polypeptide



In a particular embodiment the polypeptide of the invention is a functional polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with SEQ ID NO: 20. In particular with the mature functional polypeptide comprised in SEQ ID NO: 20. More specifically the mature functional polypeptide comprises or consists of the sequences from position 1 to 243 of SEQ ID NO: 20.
SEQ ID NO: 22 Functional Polypeptide
In a particular embodiment the polypeptide of the invention is a functional polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with SEQ ID NO: 22. In particular with the mature functional polypeptide comprised in SEQ ID NO: 22. More specifically the mature functional polypeptide comprises or consists of the sequences from position 1 to 415 of SEQ ID NO: 22.
SEQ ID NO: 24 Functional Polypeptide
In a particular embodiment the polypeptide of the invention is a functional polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with SEQ ID NO: 24. In particular with the mature functional polypeptide comprised in SEQ ID NO: 24. More specifically the mature functional polypeptide comprises or consists of the sequences from position 1 to 377 of SEQ ID NO: 24.
SEQ ID NO: 26 Functional Polypeptide
In a particular embodiment the polypeptide of the invention is a functional polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with SEQ ID NO: 26. In particular with the mature functional polypeptide comprised in SEQ ID NO: 26. More specifically the mature functional polypeptide comprises or consists of the sequences from position 1 to 259 of SEQ ID NO: 26.
SEQ ID NO: 28 Functional Polypeptide
In a particular embodiment the polypeptide of the invention is a functional polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with SEQ ID NO: 28. In particular with the mature functional polypeptide comprised in SEQ ID NO: 28. More specifically the mature functional polypeptide comprises or consists of the sequences from position 1 to 248 of SEQ ID NO: 28.
SEQ ID NO: 30 Functional Polypeptide
In a particular embodiment the polypeptide of the invention is a functional polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with SEQ ID NO: 30. In particular with the mature functional polypeptide comprised in SEQ ID NO: 30. More specifically the mature functional polypeptide comprises or consists of the sequences from position 1 to 149 of SEQ ID NO: 30.
SEQ ID NO: 32 Functional Polypeptide
In a particular embodiment the polypeptide of the invention is a functional polypeptide comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with SEQ ID NO: 32. In particular with the mature functional polypeptide comprised in SEQ ID NO: 32. More specifically the mature functional polypeptide comprises or consists of the sequences from position 1 to 202 of SEQ ID NO: 32.
SEQ ID NO: 34 Beta-glucosidase
In a particular embodiment the polypeptide of the invention is a beta-glucosidase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a beta-glucosidase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature beta-glucosidase comprised in SEQ ID NO: 34. More specifically the mature beta-glucosidase comprise or consists of the sequences from position 1 to 603 of SEQ ID NO: 34. In the present context the beta-glucosidase is defined as a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. For purposes of the present invention, beta-glucosidase activity is determined according to the basic procedure described by Venturi et al., 2002, J. Basic Microbiol. 42: 55-66, except different conditions were employed as described herein. One unit of beta-glucosidase activity is defined as 1.0 micromole of p-nitrophenol produced per minute at 50° C., pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate, 0.01% Tween-20.
SEQ ID NO: 36 Endo-arabinase
In a particular embodiment the polypeptide of the invention is a endo-arabinase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a endo-arabinase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature endo-arabinase comprised in SEQ ID NO: 36. More specifically the mature endo-arabinase comprises or consists of the sequences from position 1 to 301 of SEQ ID NO: 36. In the present context an endo-arabinase is defined as an enzyme capable of hydrolysing arabinan.
SEQ ID NO: 38 Endo-arabinase
In a particular embodiment the polypeptide of the invention is a endo-arabinase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a endo-arabinase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature endo-arabinase comprised in SEQ ID NO: 38. More specifically the mature endo-arabinase comprises or consists of the sequences from position 1 to 438 of SEQ ID NO: 38. In the present context an endo-arabinase is defined as an enzyme capable of hydrolysing arabinan.
SEQ ID NO: 40 Al Pepsin Peptidase
In a particular embodiment the polypeptide of the invention is a pepsin peptidase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a pepsin peptidase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature pepsin peptidase comprised in SEQ ID NO: 40. More specifically the mature pepsin peptidase comprises or consists of the sequences from position 1 to 396 of SEQ ID NO: 40. In the present context a pepsin peptidase is defined as an enzyme capable of hydrolysing proteins or peptides.
SEQ ID NO: 42 M43 Pepsin Peptidase
In a particular embodiment the polypeptide of the invention is a pepsin peptidase comprising or consisting of an amino acid sequence which has at least 90%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a pepsin peptidase obtainable from Botryosphaeria rhodina, in particular that strain of Botryosphaeria rhodina deposited under deposit accession number CBS 274.96, more particularly the mature pepsin peptidase comprised in SEQ ID NO: 42. More specifically the mature pepsin peptidase comprises or consists of the sequences from position 1 to 262 of SEQ ID NO: 42. In the present context a Pepsin peptidase is defined as an enzyme capable of hydrolysing proteins or peptides.
Polynucleotides
The present invention also relates to polynucleotides comprising or consisting of a nucleotide sequence encoding a polypeptide of the invention. In a particular embodiment, the nucleotide sequence is set forth in the sequences of group ADNA including nucleotide sequences differing there from by virtue of the degeneracy of the genetic code. In a further embodiment the polynucleotide of the invention is a modified nucleotide sequence which comprises or consists of a nucleotide sequence selected from the the regions of group ADNA sequences encoding a mature polypeptide and which comprises at least one modification/mutation compared with the parent nucleotide sequence comprised in the sequences of group ADNA.
The techniques used to isolate and/or clone a nucleotide sequence encoding an enzyme are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleotide sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.
The nucleotide sequence may be obtained by standard cloning procedures used in genetic engineering to relocate the nucleotide 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 fragment comprising the nucleotide 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 nucleotide sequence will be replicated. The nucleotide sequence may be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or any combinations thereof.
In particular the polynucleotide comprises, preferably consists of, a nucleotide sequence which has at least 50% identity with a nucleotide sequence selected from the regions of group ADNA sequences encoding a mature polypeptide. Particularly, the nucleotide sequence has at least 65% identity, more particularly at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with a nucleotide sequence selected from the regions of group ADNA sequences encoding a mature polypeptide. Particularly, the nucleotide sequence comprises a nucleotide sequence selected from the regions of group ADNA sequences encoding a mature polypeptide. In an even more particular embodiment, the nucleotide sequence consists of a nucleotide sequence selected from the regions of group ADNA sequences encoding a mature polypeptide.
In particular the polynucleotide comprises, preferably consists of, a nucleotide sequence encoding a mature enzyme selected from xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase and which has at least 50% identity, particularly at least 65% identity, more particularly at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with a nucleotide sequence encoding a mature enzyme selected from xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase secreted from Botryosphaeria rhodina deposited under CBS accession No. 274.96 and isolated by the present inventors.
In a particular embodiment the polynucleotide comprises, preferably consists of, a nucleotide sequence encoding a mature enzyme selected from xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase and which has at least 50% identity, particularly at least 65% identity, more particularly at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with a nucleotide sequence encoding a mature enzyme selected from from group DPolypeptide sequences.
In a particular embodiment the polynucleotide comprises, preferably consists of, a nucleotide sequence encoding a mature enzyme selected from xylanase, serine esterase, peroxidase, GH 61A polypeptide, GH 61B polypeptide, GH 61C polypeptide, GH 61D polypeptide, beta-glucosidase, endo-arabinase and pepsin peptidase and which has at least 50% identity, particularly at least 65% identity, more particularly at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with a nucleotide sequence selected from EDNA sequences
SEQ ID NO: 1
In a particular embodiment the polynucleotide of the invention encodes a GH 10 xylanase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 55 to 927 of SEQ ID NO: 1
SEQ ID NO: 3
In a particular embodiment the polynucleotide of the invention encodes a GH11 xylanase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 58 to 663 of SEQ ID NO: 3
SEQ ID NO: 5
In a particular embodiment the polynucleotide of the invention encodes a serine esterase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 55 to 1110 of SEQ ID NO: 5
SEQ ID NO: 7
In a particular embodiment the polynucleotide of the invention encodes a lipase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 55 to 1347 of SEQ ID NO: 7.
SEQ ID NO: 9
In a particular embodiment the polynucleotide of the invention encodes a peroxidase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 1 to 555 of SEQ ID NO: 9.
SEQ ID NO: 11
In a particular embodiment the polynucleotide of the invention encodes a GH61A polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 49 to 702 of SEQ ID NO: 11.
SEQ ID NO: 13
In a particular embodiment the polynucleotide of the invention encodes a GH 61 B polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 40 to 786 of SEQ ID NO: 13.
SEQ ID NO: 15
In a particular embodiment the polynucleotide of the invention encodes a GH 61C polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 55 to 819 of SEQ ID NO: 15.
SEQ ID NO: 17
In a particular embodiment the polynucleotide of the invention encodes a GH 61D polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 61 to 675 of SEQ ID NO: 17.
SEQ ID NO: 19
In a particular embodiment the polynucleotide of the invention encodes a mature functional polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 1 to 729 of SEQ ID NO: 19.
SEQ ID NO: 21
In a particular embodiment the polynucleotide of the invention encodes a mature functional polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 55 to 1299 of SEQ ID NO: 21.
SEQ ID NO: 23
In a particular embodiment the polynucleotide of the invention encodes a mature functional polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 49 to 1179 of SEQ ID NO: 23.
SEQ ID NO: 25
In a particular embodiment the polynucleotide of the invention encodes a mature functional polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 70 to 846 of SEQ ID NO: 25.
SEQ ID NO: 27
In a particular embodiment the polynucleotide of the invention encodes mature functional polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 58 to 801 of SEQ ID NO: 27.
SEQ ID NO: 29
In a particular embodiment the polynucleotide of the invention encodes a mature functional polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 157 to 603 of SEQ ID NO: 29.
SEQ ID NO: 31
In a particular embodiment the polynucleotide of the invention encodes a mature functional polypeptide and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 55 to 660 of SEQ ID NO: 31.
SEQ ID NO: 33
In a particular embodiment the polynucleotide of the invention encodes a beta-glucosidase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 46 to 1854 of SEQ ID NO: 33.
SEQ ID NO: 35
In a particular embodiment the polynucleotide of the invention encodes a endo-arabinase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 64 to 966 of SEQ ID NO: 35.
SEQ ID NO: 37
In a particular embodiment the polynucleotide of the invention encodes a endo-arabinase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 55 to 1368 of SEQ ID NO: 37.
SEQ ID NO: 39
In a particular embodiment the polynucleotide of the invention encodes a pepsin protease and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 61 to 1248 of SEQ ID NO: 39.
SEQ ID NO: 41
In a particular embodiment the polynucleotide of the invention encodes a pepsin protease and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of position 64 to 849 of SEQ ID NO: 41.
Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of a polypeptide which comprises an amino acid sequence that has at least one substitution, deletion and/or insertion as compared to an amino acid sequence selected from mature polypeptide comprised in group BPolypeptide.
It will be apparent to those skilled in the art that such modifications can be made to preserve the function of the enzyme i.e. made outside regions critical to the function of the enzyme. Amino acid residues which are essential to the function are therefore preferably not subject to modification, such as substitution. Amino acid residues essential to the function may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). Sites of substrate-enzyme interaction can be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
Moreover, a nucleotide sequence encoding an enzyme of the invention may be modified by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the enzyme encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme.
The introduction of a mutation into the nucleotide sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure, which utilizes a super coiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with Dpnl, which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA. Other procedures known in the art may also be used. For a general description of nucleotide substitution, one may consult with e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
The present invention also relates to a polynucleotide comprising, preferably consisting of, a nucleotide sequence which encodes a polypeptide of the invention and which hybridizes under high stringency conditions, preferably under very high stringency conditions with a polynucleotide probe selected from the group consisting of:

    (i) the complementary strand to a nucleotide sequence selected from the group consisting of the regions of group ADNA sequences encoding a mature polypeptide,
    (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from the group consisting of the regions of group ADNA sequences encoding a mature polypeptide,
    (iii) a fragment of (i) or (ii) encoding a secreted mature polypeptide having the function of the corresponding mature polypeptides comprised in group B Polypeptide 

(J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).



As will be understood, details and particulars concerning hybridization of the nucleotide sequences will be the same or analogous to the hybridization aspects discussed in the section titled “polypeptides of the invention” herein.
The present invention also encompasses a storage medium suitable for use in an electronic device comprising information of the amino acid sequence of polypeptides of the invention or the nucleotide sequences of the polynucleotide of the invention. The storage medium may suitably be a magnetic or optical disk and the electronic device a computing device and the information may in particular be stored on the storage medium in a digital form.
Nucleotide Constructs
The present invention also relates to nucleic acid constructs comprising a nucleotide sequence of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
A nucleotide sequence encoding an enzyme of the invention may be manipulated in a variety of ways to provide for expression of the enzyme. Manipulation of the nucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleotide sequences utilizing recombinant DNA methods are well known in the art.
The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of the nucleotide sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extra cellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof.
In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleotide sequence encoding the enzyme. Any terminator which is functional in the host cell of choice may be used in the present invention.
Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
The control sequence may also be a suitable leader sequence, a non-translated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention. Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleotide sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.
The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded enzyme into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted enzyme. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the enzyme. However, any signal peptide coding region which directs the expressed enzyme into the secretory pathway of a host cell of choice may be used in the present invention.
Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.
The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a enzyme. The resultant polypeptide may be denoted a pro-enzyme or propolypeptide. A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836.
Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences.
Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.
Recombinant Expression Vectors
The present invention also relates to recombinant expression vectors comprising the nucleic acid construct of the invention. The various nucleotide and control sequences described above may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, the nucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
The vectors of the present invention preferably contain one or more selectable markers that permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus. 
The vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the nucleotide sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleotide sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).
More than one copy of a nucleotide sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleotide sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
Recombinant Host Cells
The present invention also relates to recombinant a host cell comprising the nucleic acid construct of the invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a nucleotide sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
The host cell may be a unicellular microorganism, e.g., a prokaryote or a non-unicellular microorganism, e.g., a eukaryote.
Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In another preferred embodiment, the Bacillus cell is an alkalophilic Bacillus. 
The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).
The host cell may be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
In a preferred embodiment, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). In a more preferred embodiment, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
In an even more preferred embodiment, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
In a most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.
In another more preferred embodiment, the fungal host cell is a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
In an even more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma. 
In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In an even most preferred embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. In another most preferred embodiment, the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.
Methods for Preparing Enzyme Polypeptides
The present invention also relates to methods for producing an enzyme of the invention comprising (a) cultivating a strain comprising a nucleotide sequence encoding an enzyme of the invention which strain is capable of expressing and secreting the enzyme and (b) recovering the enzyme. In a particular embodiment the strain is a wild type strain such as the Botryospaeria rhodina CBS 274.96, while in another embodiment the strain is a recombinant host cell as described, supra.
In these methods of the invention, the cells are cultivated in a nutrient medium suitable for production of the enzyme 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. 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). As the enzyme is secreted into the nutrient medium, the enzyme can be recovered directly from the medium.
The resulting enzyme may be recovered by methods known in the art. For example, the enzyme may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. 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), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
Transgenic Plants
The present invention also relates to a transgenic plant, plant part, or plant cell that has been transformed with a nucleotide sequence encoding an enzyme of the invention so as to express and produce the enzyme. In one embodiment the plant could be used as host for production of enzyme in recoverable quantities. The enzyme may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant enzyme may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor. In particular the plant or plant parts expressing the enzyme may be used as an improved starting material for production of fuel-alcohols or bio-ethanol
The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana. 
Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers. Also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part.
Also included within the scope of the present invention are the progeny of such plants, plant parts and plant cells.
The transgenic plant or plant cell expressing an enzyme of the invention may be constructed in accordance with methods known in the art. Briefly, the plant or plant cell is constructed by incorporating one or more expression constructs encoding an enzyme of the invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
Conveniently, the expression construct is a nucleic acid construct which comprises a nucleotide sequence encoding an enzyme of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleotide sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).
The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the enzyme is desired to be expressed. For instance, the expression of the gene encoding an enzyme of the invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.
For constitutive expression, the 35S-CaMV promoter may be used (Franck et al., 1980, Cell 21: 285-294). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22: 573-588).
A promoter enhancer element may also be used to achieve higher expression of the enzyme of the invention in the plant. For instance, the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding an enzyme of the present invention. For instance, Xu et al., 1993, supra disclose the use of the first intron of the rice actin 1 gene to enhance expression.
The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).
Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38). However, it can also be used for transforming monocots, although other transformation methods are generally preferred for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428.
Following transformation, the transformants having incorporated therein the expression construct are selected and regenerated into whole plants according to methods well known in the art.
The present invention also relates to methods for producing an enzyme of the invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleotide sequence encoding an enzyme of the invention under conditions conducive for production of the enzyme and (b) recovering the enzyme.
Compositions Comprising Enzyme Polypeptides and Methods for Their Preparation
The invention provide a composition comprising a polypeptide of the invention and an excipient and a method for preparing such a composition comprising admixing the polypeptide of the invention with an excipient. In a particular embodiment the polypeptide of the invention is the major (polypeptide) component of the composition, e.g., a mono-component composition. The excipient in this context is to be understood as any auxilliary agent or compound used to formulate the composition and includes solvent, carriers, stabilizers and the like.
The composition may further comprise one or more additional enzymes, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a solid composition. For instance, the enzyme composition may be formulated using methods known to the art of formulating polypeptides and/or pharmaceutical products, e.g. into coated or uncoated granules or micro-granules. The polypeptide of the invention may thus be provided in the form of a granule, preferably a non-dusting granule, a liquid, in particular a stabilized liquid, a slurry or a protected polypeptide. For certain applications, immobilization of the polypeptide on a solid matrix may be preferred.
The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art e.g. by stabilizing the polypeptide in the composition by adding and antioxidant or reducing agent to limit oxidation of the polypeptide or it may be stabilized by adding polymers such as PVP, PVA, PEG or other suitable polymers known to be beneficial to the stability of polypeptides in solid or liquid compositions.
In a further embodiment the composition of the invention is a detergent composition which, in addition to the polypeptide of the invention, comprises a surfactant and optionally compounds selected from the group consisting of builders such as zeolites, bleaching agents such as percarbonate, bleach enhancers such as TAED or NOBS, suds suppressors, fragrants, etc.
In a further embodiment the composition of the invention is a feed composition that in addition to the polypeptide of the invention comprises a cereal or grain product.
In a further embodiment the composition of the invention is a food composition such as a baker's flour composition, a brewed product, a fruit juice, an oil or lard product comprising the polypeptide of the invention.
In a further embodiment the composition of the invention is a pulping composition, which in addition to the polypeptide of the invention, comprises pulp.
In a further embodiment the composition of the invention is a biocidal composition, which comprises in addition to the polypeptide of the invention, an oxidoreductase enhancer.
Use of Enzyme Polypeptides or Compositions Comprising them
In still further aspects the invention provides use of the polypeptides or polynucleotides of the invention or a composition comprising said polypeptides or polynucleotides in various applications, particularly (technical) processes such as processes performed in industry or household, herein under for commercial research purposes. Hence the invention encompasses a process comprising employing a polypeptide of the invention or a polynucleotide of the invention in a (technical) industrial, research or household process.
In one embodiment the polypeptide or the composition of the invention is used for cleaning a cellulosic fabric.
In another embodiment the polypeptide or the composition of the invention is used to prepare a food or feed additive.
In yet another embodiment the polypeptide or the composition of the invention is used for treatment of lignolosic materials and pulp.
Detergent Disclosure
The polypeptide of the invention may be added to and thus become a component of a detergent composition.
The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
In a specific aspect, the invention provides a detergent additive comprising the polypeptide of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with the specific position substitutions mentioned therein.
Preferred commercially available protease enzymes include Alcalase®, Savinase®, Primase®, Duralase®, Esperase®, and Kannase® (Novozymes A/S), Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect OxP®, FN2®, and FN3® (Genencor International Inc.).
Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
Preferred commercially available lipase enzymes include LipolaseTM, Lipolase UltraTM and LipexTM (Novozymes A/S).
Amylases: Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1,296,839.
Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with the specific position substitutions mentioned therein.
Commercially available amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (from Genencor International Inc.).
Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
Commercially available cellulases include Celluzyme®, and Carezyme® (Novozymes), Clazinase®, and Puradax HA® (Genencor International Inc.), and KAC-500(B) ® (Kao Corporation).
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
Commercially available peroxidases include Guardzyme® (Novozymes A/S).
The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granule, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.
The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.
The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.
When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).
The detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
The detergent may contain a bleaching system which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
It is at present contemplated that in the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per litre of wash liquor.
The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 that is hereby incorporated as reference.
Deposited Microorganism
The following microorganism was deposited by the applicant according to the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for the Purpose of Patent Procedures at Centraalbureau voor Schimmelcultures, Fungal and Yeast Collection, Uppsalalaan 8, 3584 CT Utrecht, P.O.Box 85167, 3508 AD Utrecht, The Netherlands:













Name:
Botryosphaeria rhodina


Synonym:
Diplodia gossypina (SBL 274)



(Berkeley & M. A. Curtis) von Arx



Protolog Arx, J. A. von 1970, “The genera



of fungi sporulating in pure culture”: 143


Classification:
Dothideaceae, Dothideales, Dothidemycetes;



Ascomycota


Deposit accession number:
CBS 274.96


Date of deposit:
12-Mar-1996









EXAMPLES
Example 1
Identifying Functional Polypeptides Secreted by Botryosphaeria rhodina CBS 274.96
Enzyme Finger Printing of Culture Fluids
An enzyme activity profile was obtained by assaying the culture broth on a wide spectrum of enzyme assays. 96 wells microtitre (MT) plates were prepared with substrates and stored at +10° C. until use. Two different pH varieties were prepared: pH3 and pH7. Following substrates were used: 0.05% AZCL (Mazurine dyed and cross-linked substrates, Megazyme)—Amylose, Arabinan, Beta Glucan (Barley), Casein, Collagen, Curdlan, Dextran, Galactan (potato), Galactomannan (Carob), He-Cellulose, Pullulan, Xylan (oat), and Xyloglucan (AZCL-casein could not be used at pH3, and was therefore left out from these plates).
Preparation of pH3 Substrates:


    0.1 g of each AZCL substrate was dissolved in 100 ml 0.2M Succinic acid pH3+10 microlitres TritonX-100 (0.01%), to give a final concentration of 0.1% AZCL.

Preparation of pH7 Substrates:

    0.1 g of each AZCL substrate was dissolved in 50 ml sterile H20 plus 10 microlitres TritonX-100 (0.01%). 50 ml 0.4M MOPS pH 7 was added to each 50 ml AZCL substrate, to give a final volume of 100 ml and a final concentration of 0.2M buffer, 0.1% AZCL.


Laccase and lipase activity assays were included and substrates were prepared as follows:
Preparation of Laccase Substrate:


    35 ml 0.08 mg/ml Chicago Sky Blue in 0.2 M phosphate/borate-buffer, pH 9, was prepared

Preparation of Lipase Substrate:

    A polyvinyl alcohol (PVA)/soy bean oil emulsion was prepared by mixing a 2% PVA solution with soy bean oil 3:1. The oil was emulsified using ULTRA-TURRAX mixer and 12 ml of the emulsion was mixed with 500 ml 0.2M sodium-acetate buffer including 10mM CaCl2 pH 5.5 and 5 ml 0.2% Crystal Violet solution.


US Sterilin 96-wells MT plates were used with a Multidrop S20 Stacker, Titertek Instruments, Inc., Alabama. 200 microlitres of each AZCL-substrate and of the lipase substrate and 150 microlitres of the laccase substrate were dispensed into MT wells and 30-50 μl culture broth were added to each substrate and incubated over night at 26 degree celcius. Result scores were made the assays as follows: 0: no activity, 1: weak activity, 2: strong activity.










Result table I: Fingerprinting of D. gossypina culture broth












Substrate
Enzyme activity
pH 3
pH7







AZCL-Amylose
α-amylase
0a
1



AZCL-Arabinan
endo-1,5-α-L-
0
2



(Debranched)
arabinanase



AZCL-Beta-Glucan
β-glucanase,
2
1



(Barley)
lichenase and




cellulases



AZCL-Casein
proteases
Ntb
2



AZCL-Collagen
proteases
0
1



AZCL-Curdlan
endo-1,3-β-D-
1
0




glucanase



AZCL-Dextran
endo-1,6-α-D-
0
0




glucanase




(dextranase)



AZCL-Galactan
endo-1,4-β-D-
0
2



(Potato)
galactanase



AZCL-
endo-1,4-β-D-
2
2



Galactomannan
mannanase



(Carob)



AZCL-HE-Cellulose
endo-cellulase
2
2



AZCL-Pullulan
limit-dextrinase
0
0




(pullulanase)



AZCL-Xylan (Oat
endo-1,4-β-D-
2
2



Spelts)
xylanase



AZCL-Xyloglucan
endo-cellulases
0
2



Chicago Sky Blue
Laccase
0
0



PVA/soyabean oil
Lipase
0
0







aresult score for the activity assays: 0 = no activity, 1 = weak reaction and 2 = strong reaction



bnt = not tested






A. cDNA Library Construction

cDNA from Botryosphaeria rhodina CBS 274.96 was prepared by using standard molecular biology techniques (Ausuble et al. 1995 “Current protocols in molecular biology” Publ.:John Wiley and sons).
Fermentation of the biomass used in the cDNA library production was initiated from an inoculated PDA plate that had been incubated for 7 days at 28 degrees. Several mycelia-PDA agar plugs were inoculated in shake flasks with Mex1 media containing (per litre): 20 g of soy bean, 15 g of wheat bran, 10 g of cellulose Avicel, 5 g of maltodextrin 01, 3 g of bacto peptone, 0.2 g of pluronic PE6100, and 1 gram Olive oil. The flasks were shaken at 150 RPM at 26 degrees. After 7 days the mycelium was harvested by filtration through Miracloth and frozen in liquid nitrogen and stored at −80° C. until use.
RNA isolation: The total RNA was prepared from frozen, powdered mycelium of Botryospaeria rhodina by extraction with guanidium thiocyanate followed by ultracentrifugation though a 5.7M CsCl cushion (Chirgwin, J. M., Przbyla, A. E., Macdonlad, R. J., and Ruttwer W. J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry 18, 5294-5299, 1979). The polyA enriched RNA was isolated by oligo (dT)-cellulose affinity chromatography (Aviv, H., and Leder, P., Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose, Proc. Natl. Acad. Sci. USA 69 (6), 1408-1412, 1972).
Construction of the cDNA library: Double stranded cDNA was synthesized according to the general methods of Gubler U. and Hoffman, B. J., A simple and very efficient method for generating cDNA libraries, Gene 25(2-3), 263-269, (1983); Sambrook, J., Fritsch, E. F., and Maniantis, T. Molecular cloning: A laboratory Manual, 2nd ed., 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Kofod et al. (1994) using a polyA-Not1 primer (Promega, USA). After synthesis, the cDNA was treated with mung bean nuclease, blunt ended with T4 DNA polymerase, and ligated to a 50 fold molar excess of EcoRI adaptors (Invitrogen, USA). The cDNA was cleaved with NotI restriction enzyme (New England Biolabs, USA) according to the manufacturer's instructions and the cDNA size fractionated by agarose gel electrophoresis. cDNA corresponding to 700 bp and larger were excised from the gel and purified using the GFX DNA isolation kit (AP Biotech). The prepared cDNA was then directionally cloned by ligation into EcoRI-NotI cleaved pMHas5. The ligation mixture was electroporated into DH10B cells (Invitrogen) and plated on LB agar with 50 mgs/liter kanamycin. A cDNA plasmid pool was prepared from 30,000 total transformants of the original cDNA-pMHas5 vector ligation. Plasmid DNA was prepared directly from a pool of colonies recovered from solid LB kanamycin selective media according to the Qiagen protocol for plasmid DNA isolation (Qiagen Inc. GMBH).
Vector used for cloning was pMhas5, which is described in patent WO 03/044049 and has the following features:















Feature
Location
Description









CDS
 365-1156
Kanamycin resistance



CDS
2232-2387
Beta galactosidase alpha peptide



−10 signal
2189-2192
Shine Dalgarno



promotor
2101-2189
Lac promotor



misc feature
626-650
KanP1 primer for BACE system










Notable features of this plasmid are the EcoRI-NotI restriction sites proximal to the Shine Dalgarno region of the Lac promoter. This allows EcoRI-NotI adapted cDNAs to be cloned into the vector and the resulting constructs to be actively transcribed and translated in the E. coli host.

B. Transposon Construction and Preparation

The rationale behind the methology of Transposon Assisted Signal Trapping (TAST) as described in WO 01/77315 Al is to fuse all genes within a selected genome with a gene encoding a signalless beta-lactamase via a transposon tag. Hence when growing host cell clones comprising the genes of a genome fused with a gene encoding a signalless beta-lactamase via a transposon tag in an ampicillin containing medium only those clones expressing and secreting a beta-lactamase will survive. However the beta-lactamase will only be secreted if the gene to which the beta-lactamase gene is fused has an intact promotor and ribosome binding site (i.e. a gene which is expressed by the cell to produce a polypeptide in real life), which can be recognized in the host strain, and if the beta-lactamase is translated so that the synthesized polypeptide is transported across the cytoplasma membrane and folded correctly. Hence, when inserting the fused gene into a selected host cell, those clones, which are ampicillin resistant contains a gene which encodes a functional secreted polypeptide.
Usually, when employing the TAST methodology it is even not necessary to express the entire gene. When tagging the genes with a transposon, expression of the N-terminal part of the genes as protein fusion shows that the genes contain intact transcription, translation and secretion sequences. Hence expression of the N-terminal part of the genes as protein fusion is usually regarded as sufficient for assuring expression and secretion of the entire gene.
Thus it can be concluded that the genes obtained by the TAST method actually do encode secreted functional polypeptides.
Construction of a SigA4 Transposon Containing the β-lactamase Reporter Gene:


    Following the instructions of WO 01/77315 Al, the construction of a transposon containing a signal-less β-lactamase gene was carried out using standard molecular biology techniques. The signal-less β-lactamase gene was initially PCR amplified from the vector pUC19) using a proofreading polymerase (Pfu Turbo, Stratagene, USA). The resulting PCR fragment contained the restriction sites NotI and EcoRI in order to aid cloning. The plasmid pEntranceposon(Camr) containing the Entranceposon and the antibiotic resistance markers CAT (encoding chloramphencol resistance in the transposon) was obtained from Finnzymes, OY (Espoo Finland). The plasmid was digested with the restriction enzymes NotI and EcoRI, gel purified and ligated with the signal-less β-lactamase containing fragment. The ligation was transformed into electro-competent DH10 B cells and the E. coli clone containing the recombinant plasmid with the signal-less β-lactamase was identified by restriction analysis and named SigA2.


For transposon preparation, a smaller derivative of SigA2 was constructed, which lacked the bla gene encoding beta-lactamase: Two oligonucleotide primers SigA2NotU-P 5′-TCG CGA TCC GTT TTC GCA TTT ATC GTG AAA CGC T-3′ (SEQ ID NO: 43) and SigA2NotD-P 5′-CCG CAA ACG CTG GTG AAA GTA AAA GAT GCT GAA-3′ (SEQ ID NO: 44), which bind to the start and stop of the bla gene of SigA2 directing outwards were used PCR amplify SigA2 without the bla gene. A amplificate of approx. 3.6 kb generated in the this PCR reaction was relegated and transformed in to a suitable E. coli strain. A plasmid of 3.6 kb was isolated from a transformant which was able to grow on LB chloramphenicol but not on LB ampicillin. This plasmid maintained both BgIII sites and lacks the active bla gene and was called pSig4.
60 microlitres of pSigA4 plasmid DNA preparation with a concentration of 0.3 μg/μl was digested with BgIII and separated on an agarose gel. The SigA2 transposon DNA band of 2 kb was eluted and purified by using the “GFXTMPCR, DNA and Gel Band Purification Kit” (Amersham Pharmacia Biotech Inc, USA) according to the instructions of the vendor and eluted in 200 microlitres EB buffer. SigA2 prepared in this manner could be be used for transposon assisted signal trapping (TAST vide infra).

C. Transposon Tagging

The transposon prepared from pSigA4 carries a 5′-truncated bla-gene encoding a β-lactamase from which the secretion signal was removed. The β-lactamase conveys ampicillin resistance on E. coli only when the protein is secreted to the periplasm, whereas cytoplasmic expression of β-lactamase does not confer ampicillin resistance. Without a signal sequence, the β-lactamase enzyme will not be transported to the periplasm and therefore the clone will not grow on media containing ampicillin. The signal-less β-lactamase gene was contained within the transposon in such a way that there was a continuous open reading frame between the transposon border and the β-lactamase coding region. In this way the modified transposon, when it transposes into a gene encoding a protein that is secreted, could cause an in-frame fusion with the target gene. This resulted in a fusion gene product that is secreted to the periplasm of E. coli and conveys resistance to the ampicillin. If the transposon integrated even in-frame into a gene encoding a non-secreted protein, the respective host will not become ampicillin resistance.
D. Transposon Assisted Signal Trapping of Botryosphaeria rhodina 
A complete description of transposon assisted signal trapping can be found in WO 01/77315. A cDNA plasmid pool was prepared from 30,000 total transformants of the original cDNA-pMHas5 vector ligation. Plasmid DNA was prepared directly from a pool of colonies recovered from solid LB selective media according to the Qiagen protocol for plasmid DNA isolation (Qiagen Inc.). The plasmid pool was treated with transposon SigA2 and MuA transposase according to the transposase manufacturer's instructions (Finnizyme, Finland).
For the in vitro transposon tagging of the Botryosphaeria rhodina CBS 274.96 cDNA library, 4 or 8 microlitres of SigA2 transposon containing approx. 2.6 micrograms DNA were mixed with 1 microlitres of the DNA concentration of the plasmid pool DNA of the Botryosphaeria rhodina CBS 274.96 cDNA library, 2 microlitres of Finnzymes MuA Transposase (0.22 micrograms/microlitre) and 5 microlitres of 5× buffer from Finnzymes O Y, Espoo, Finland) in a total volume of 50 microlitres and incubated at 30° C. for 3.5 hours and followed by heat inactivation at 75° C. for 10 min. The DNA was precipitated by addition of 5 microlitres 3M Na-acetate pH 5 and 110 microlitres 96% ethanol and centrifugation for 30 min at 20000 rpm. The pellet was washed and dried and resuspended in 10 microlitres TE buffer.
1.5 microliter of the transposon tagged plasmid pool were electroporated into 20 microliter DH10B ultra-competent cells according to the standard protocol provided with the cells (Gibco-BRL) in a Biorad Gene Pulse device (50 uF, 25 mAmp, 1.8 kV)
Electroporated cells were incubated in SOC media with shaking (28 degrees celcius, 2 hours, 250 rpm) before being plated on selective media. Three agar media were used:

    LB+50 microgram pr. ml kanamycin,
    LB+kanamycin+15 microgram pr. ml chloramphencol, and/or
    LB+kanamycin+chloramphenicol+12.5 microgram pr. ml ampicillin.


From dilution plating of the electroporation onto LB+kanamycin+chloramphenicol media, it was determined that approximately 72.000 colonies were present containing a cDNA library plasmid with a SigA2 transposition per electroporation and that approximately 69 colonies were recovered under triple selection (LB, kanamycin, chorlamphenicol, ampicillin). Further electroporation and plating experiments were performed until 445 colonies, in all, were recovered from the experiment under triple selection. The colonies were miniprepped according to the Qiagen Qiaturbo96 protocol (Qiagen Inc.—USA). Plasmids were sequenced with the transposon forward and reverse primers (primers A and B) according to the procedure disclosed in the examples of international patent application WO 01/77315 (page 28)










Primer A:




AGCGT TTGCG GCCGC GATCC
(SEQ ID NO: 45)



 



Primer B:



TTATT CGGTC GAAAA GGATC C
(SEQ ID NO: 46)






E. Sequence Assembly and Annotation

DNA sequence was obtained for the reactions on an AB3700 capillary sequencer. Sequences were trimmed to remove vector and transposon sequence and the A and B primer reads for each plasmid. This resulted in 225 assembled sequences which were grouped into 148 contigs by using the program PhredPhrap (Brent Ewing, LaDeana Hillier, Michael C. Wendl, and Phil Green; Base-calling of automated sequencer traces using phred I. Accuracy assessment; Genome Research; 8:175-185; 1998; Brent Ewing and Phil Green; Base-calling of automated sequencer traces using phred II. Error probabilities; Genome Research 8:186-194; 1998). All 148 contigs were subsequently compared to sequences available in standard public DNA and protein sequences databases (TrEMBL, SWALL, PDB, EnsemblPep, GeneSeqP) by using the program BLASTX 2.0a19MP-WashU [14Jul. 1998] [Build linux-x86 18:51:44 30 Jul. 1998] (Gish, Warren 1994-1997—Unpublished; Gish, Warren and David J. States; Identification of protein coding regions by database similarity search; Nat. Genet. 3:266-72; 1993).
The obtained sequences being, the majority of which were functional genes encoding intact and functional polypeptides, by being obtained from ampicillin resistant clones as explained supra, were used as a basis for the manual analysis.
In order to verify that ampicillin resistance of the colonies were from increased beta lactamase activity, beta-lactamase activity was tested in ten different signal trapped clones with different transposon landing sites on the cDNA. Plasmid DNA was re-transformed into One Shot TOP10 chemically competent E. coli cells according to the protocol of Invitrogen Life Technologies and the transformation mixture spread on LB agar with kanamycin (50 μg/ml) and chloramphenicol (10 μg/ml). Colonies were replica plated on LB kanamycin, chloramphenicol with 15 (10 μg/ml) ampicillin after 2 days at 28 degrees C. After 3 days at 28 degrees C., the true growers were inoculated into 10 ml LB medium with Kanamycin (50 μg/ml) and chloramphenicol (10 μg/ml), and incubated over night at 37 degrees C., 275 rpm. The overnight culture were diluted 1:100 in fresh LB Kan/CAM medium and grown to OD600=0.5. The cells were harvested by centrifugation and washed once in 0.9% NaCl. Pellet were suspended in sonication buffer (100 mM Tris-HCl, 2 mM EDTA, pH 8.0), the number of cells adjusted to an optical density of OD600=0.5 and sonicated at an of amplitude 1.5 microns using a Soniprep 150, (MSE). The samples were centrifuged at 15.000 rpm for 10 min. and the supernatant used for beta-lactamase assay. The supernatant was mixed with 50 μl nitrocefin chromogenic beta-lactamase substrate. (1 mg/ml in 50% DMSO; 0.05 M PO4 buffer) and 0.1 M PO4 buffer, pH 7.0 up to 1 ml in a plastic cuvette and the change in Abs482 was measured in an Ultrospec 3300 pro spectrophotometer (O'Callahan, 1972). The results were as follows:







TABLE IX







β-lactamase activity of clones with different distances between


the Shine Dalgarno (SD) sequence and the ATG translation


initiation codon and transposon landing site.













β-lactamase





activity



Transposon landing
Distances between SD
(Normalized to



site on the cDNA
sequence and the ATG
trappant


Clones
(bp)
initiation codon (bp)
ZY063807)













ZY063832
806
102
5.5


ZY063832
650
102
11.5


ZY063836
991
35
35.5


ZY063836
991
35
4.0


ZY063807
851
256
1.0


ZY063827
568
8
202.0


ZY063816
476
126
16.0


ZY063816
634
126
3.0


ZY065171
469
9
106.5


ZY065171
684
9
52.0









The table above illustrates three main points:

    1) Beta-lactamase activity can be detected in all ten clones. It should be emphasized that corresponding plasmid containing E. coli transformants are resistant to 50 mg/liter ampicillin selection. Combining these two facts, indicates that all ten clones produce an hybrid protein consisting of part of the transposon tagged cDNA fused to the beta lactamase gene in such a manner as to provide an peptide with beta lactamase activity.
    2) Because the cDNA of the ten clones were completely sequenced, the landing site of the SigA2 transposon in each clone was established. The 10 clones all had a SigA2 transposon in the correct orientation and in the correct reading frame to promote a hybrid fusion between the N terminal of the native encoded protein and the transposon encoded beta lactamase.
    3) In Eukaryotic cDNAs, untranslated upstream (5′ UTR) leaders can vary in length from 1 bp to several hundred base pairs. For the cDNA to be optimally translated from the E. coli translation elements such as the Shine-Dalgarno region, an optimal distance of between 4 and 11 base pairs before the ATG start codon is optimal. In the table, we can see that 5′UTRs much longer than optimal still allow for some expression of the cDNA hybrid-beta lactamase fusion protein because beta lactamase activity is observed from the transformed E. coli in the example.


The obtained nucleotide sequences of the invention are functional genes which encode intact and functional polypeptides, not only for the reasons above, but because they were obtained from ampicillin resistant clone. The clones obtained herein were ampicillin resistant, because after transposon tagging, the genes were fused with the signalless beta-lactamase gene, which is only expressed when fused to a gene with an intact promoter and ribosome-binding site, which can be recognized in the host strain, and translated and transported across the cytoplasm membrane and folded correctly.
Therefore is could be concluded that the genes of the invention actually do encode active secreted polypeptides. For the 16 genes sequence analysis revealed, that in-frame fusion with the signalless beta-lactamase gene was obtained. In addition, the intactness of the genes' open reading frame was confirmed by determining the entire nucleotide sequence.
Example 2
Determining Enzyme Function by Homology
The function of a gene or the encoded polypeptide can be predicted by sequences comparison with genes or polypeptides of known function. Similarities between group ADNA and group Bpolypeptide sequences and sequences from public and internal databases were analysed, to determine the functionality of the group ADNA and group Bpolypeptide sequences. The sequence comparison was carried out using the program BLASTX 2.0a19MP-WashU [14 Jul. 1998]. A careful manual analysis of sequence alignments of group ADNA and group Bpolypeptide sequences to their closest related sequences with known function made it possible to predict the function of these genes and the encoded polypeptides. Even when the overall amino acid identity was below 40%, which can make it difficult to make reliable predictions, it was possible to predict the function of group ADNA and group Bpolypeptide sequences by carefully analysing and interpreting the amino acid residues in the catalytic sites and/or in important regions of the polypeptide sequences. If the amino acids of the catalytic site of known sequences were also present in the polypeptide of the invention, combined with a sufficient overall amino acid identity, it was concluded that the polypeptide from Botryosphaeria rhodina CBS 274.96 had the same function as the known sequence.
Example 3
Preparing Group Bpolypeptide Polypeptides
To prepare polypeptides from cDNA sequences of a filamentous fungus such as Botryosphaeria rhodina CBS 274.96, it was feasible to express the cDNA in yeast or another filamentous fungus. A good choice, serving as a non-exhaustive example, is expression in Aspergillus oryzae. The example given below is how a cDNA encoding a xylanase (SEQ ID No: 3) was expressed in A. oryzae. 
An expression plasmid pDaU71 was used. This plasmid contained (1) the A. nidulans amdS gene as selection marker in Aspergillus, (2) the yeast URA3 gene, interrupted by the ampicillin resistance gene for selection in E. coli, (3) a duplication of the A. niger NA2 promoter (neutral amylase) with 3 extra amyR-sites+ the 5′ untranslated part of the A. nidulans TPI promoter for heterologous expression and (4) the A. niger AMG terminator. As template for amplication of the relevant portion of the Botryosphaeria rhodina cDNA library pool, the following primers were used in the PCR reaction:








Primer #166:



CGCGGATCCACCATGGTCTCCTTCAAGTCGATTC
(SEQ ID NO: 47)


 


Primer #167:


CCGCTCGAGTTACTGCACGGTAATCGTAGC
(SEQ ID NO: 47)






The following conditions were used:

    Extensor Hi Fidelity Reddy Load DNA polymerase PCR mix was used according to the manufacturer's instructions (ABGene, Great Britain).



















cDNA plasmid pool (1 nanogram/microlitre):
5
microlitre



Primer #166
1
microlitre



Primer #167
1
microlitre



PCR master mix
12.5
microlitre



Deionised water
7.5
microlitre



Total volume
25
microlitre










The reaction was transferred to an MJ Research DNA engine prewarmed to 94° C. and the following cycle was performed:
















94° C.
 2 minutes



then 25 cycles of



94° C.
30 seconds



53° C.
30 seconds



72° C.
 1 minute



then



72° C.
10 minutes.










Five microlitre of product was analyzed on a 1% agarose gel to confirm the correct size and quantify the amount of PCR product produced. The remaining 20 microlitre mixture was GFX purified according to the manufacturer's instructions (AP Pharma). 20 microlitre of the 40 microlitre purified product was used for a standard BamHI-XhoI restriction digest in a 30 microlitre standard overnight digestion reaction. The restricted product was once again purified by GFX and used in a standard ligation recation with BamHI-XhoI restricted and purifed pDau71. The ligation product was transformed into DH10B E. coli cells (E. coli DH10B or TOP10 (available from Invitrogen) could be used as cloning hosts in construction of the expression vector) and plated on LB ampicillin media. Ten transformants were selected for plasmid DNA purification and were sequenced to confirm the sequence integrity of the insert. One PCR error free clone pPFJo147 was selected for further studies.

Aspergillus oryzae strain BECh2, which was constructed as described in WO 00/39322 (BECh2 is derived from strain Aspergillus oryzae JaL228, which is constructed on the basis of the deposited strain Aspergillus oryzae IFO 4177 as described in WO 98/12300). Transformation and culture conditions were performed according to Christiansen et al., 1988, Biotechnology 6, 1419-1422 and as described in WO 01/12794-A page 63. After one round of reisolation, 10 ml YPglucose or YPmaltose medium in Nunc tubes were inoculated with spores from the transformants and the cultures were inoculated for 3 days at 30° C.
10 microlitre supernatant samples from the above described 10 ml cultures were subjected to SDS-gel electrophoresis. The gel was stained with SYPRO Orange Protein Gel Stain (Molecular Probes). Several Aspergillus transformants had a prominent band on the SDS gel. These positives were further analyzed for xylanase activity by performing an AZCL-wheat arabinoxylan assay at pH 6.0. The substrate, AZCL-Arabinoxylan from wheat (Megazyme), was prepared as a 0.2% w/v suspension in 0.2 M Na-phosphate buffer pH 6.0 +0.01% Triton-X 100. 900 microlitre substrate was preheated to 37° C. in an Eppendorf thermomixer. 100 microlitre crude culture fluid supernatant from the recombinant host strain Bech2 or the different samples was added to the substrate and incubated for 15 min at 37° C. at maximum speed. The reaction mixture was then placed on ice for 2 minutes and then centrifuged for 1 min 20.000×G. 2×200 microlitre supernatant was transferred to a microtitter plate and measured at OD 590. Activity was determined as an increase in absorbance.
Example 4
Determining Xylanase Activity
The culture fluid or a cell lysate of a host strain synthesising and secreting a xylanase in a suitable buffer is used for measuring the activity. A suitable volume of such a sample is spotted on agarose plates which contain the insoluble chromogenic substrate AZCL-Birch xylan (MegazymeTM) and a suitable buffer at pH, e.g. pH is 4.5-7.5. The plate is incubated for an appropriate time, e.g. one day, at an appropriate temperature, e.g. 37□C. The activity is visible as blue halos around the spots.
Example 5
Determining Peroxidase Activity
Peroxidase can be determined spectrophotmetricly using the 2,4 dichlorophenol method of Ishida et al., 1987: Ishida, A., N. Futamura, and T. Matsusaka. 1987. Detection of peroxidase activity and its localisation in the forespore envelopes of Bacillus cereus. J. Gen. Appl. Microbiol. 33:27-32. As indicator a mixture of 1.0 mM 2.4 Dichlorophenol and 82 mM 4-aminoantipyrene in 100mM potassium phosphate buffer (pH 7.0) can be used while a substrate of hydrogen peroxide (Sigma), 50 mM in 100 mM potassium phosphate buffer (pH7.0) is suitable.
200 microlitre of appropriately diluted culture fluid supernatant is added to a 1 ml plastic cuvette. 200 microlitre of the indicator mixture is added. Reactions are initiated by the addition of 200 microlitre hydrogen peroxide substrate. Changes in absorbance are observed in a standard spectorophotometer measuring at a wavelength of 510 nm.
Example 6
Determining Cellulose Degradation Boosting Effect on Cellulose Degrading Enzymes or Enzyme Mixtures
Some secreted proteins demonstrate synergistic action in degradation of cellose in the presence of cellulose degrading enzymes or mixtures thereof. Such secreted proteins may or may not have, in themselves, hydrolase activity. Examples of such secreted proteins and how one detects their cellulose degradation boosting effect can be found in patent application no. U.S. Pat. No. 11/046,124 and corresponding PCT application PCT/US2005/003525 published 30 Jun. 2005, in particular using the examples 24 and anyone from examples 25 to 28, hereby incorporated by reference.
One way of determining this boosting effect is as follows: Culture fluid or a cell lysate of a host strain synthesising and secreting a cellulase boosting polypeptide are concentrated using an Amicon stirred cell equipped with a PM10 membrane, 10 kDa cutoff (Millipore, Billerica, Mass.), and desalted using an Econo-Pac 10DG column (BioRad Laboratories, Hercules, Calif.). After assay of the protein concentration by BCA (bicinchoninic acid, P. K. Smith et al., 1985, Anal. Biochem. 150: 76) Protein Assay Kit (Pierce, Rockford, Ill.) using BSA as standard these polypeptide stocks are stored at −20° C. The polypeptides are not further purified, and stocks are added to reaction mixtures based on total protein measured.
Corn stover is pretreated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL) using dilute sulfuric acid. The following conditions are used for the pretreatment: 1.4 wt % sulfuric acid at 165° C. and 107 psi for 8 minutes. According to NREL, the water-insoluble solids in the pretreated corn stover (PCS) contain 56.5% cellulose, 4.6% hemicellulose and 28.4% lignin. Cellulose and hemicellulose are determined by a two-stage sulfuric acid hydrolysis with subsequent analysis of sugars by high performance liquid chromatography using NREL Standard Analytical Procedure #002. Lignin is determined gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid using NREL Standard Analytical Procedure #003. Prior to enzymatic hydrolysis, the PCS is ished with a large volume of DDI water on a glass filter; finding the dry weight of the water-ished PCS. Milled PCS is prepared from the water-ished PCS by milling in a coffee-grinder and subsequent ishing with deionized water on a 22 μm Millipore Filter (6P Express Membrane, Stericup, Millipore, Bedford, Mass.).
Hydrolysis of PCS is conducted using 1.1 ml Immunoware microtubes (Pierce, Rockford, Ill.) using a total reaction volume of 1.0 ml. In this protocol hydrolysis of PCS (10 mg/ml in 50 mM sodium acetate pH 5.0 buffer) is performed using different protein loadings (expressed as mg of enzyme per gram of PCS) of a test polypeptide of the invention or Celluclast® 1.5 L sample (Novozymes A/S, Bagsvaerd, Denmark) in the presence of 3% Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) of the cellulase protein loading. Screening of polypeptides of the invention for PCS hydrolyzing capability is performed at 50° C. (Isotemp 102S water baths or TS Autoflow CO2 Jacketed Incubator). Typically, reactions are run in quadruplicate and aliquots taken during the course of hydrolysis. PCS hydrolysis reactions are stopped by mixing a 20 μl aliquot of each hydrolyzate with 180 μl of 0.11 M NaOH (stop reagent). Appropriate serial dilutions are generated for each sample and the reducing sugar content determined using a para-hydroxybenzoic acid hydrazide (PHBAH, Sigma, St. Louis, Mo.) assay adapted to a 96 well microplate format as described below. Briefly, a 90 μl aliquot of an appropriately diluted sample is placed in a 96 well conical bottomed microplate. Reactions are initiated by adding 60 μl of 1.5% (w/v) PHBAH in 2% NaOH to each well. Plates are heated uncovered at 95° C. for 10 minutes. Plates are allowed to cool to room temperature (RT) and 50 μl of distilled H2O added to each well. A 100 μl aliquot from each well is transferred to a flat bottomed 96 well plate and the absorbance at A510nm measured using a SpectraMax Microplate Reader (Molecular Devices, Sunnyvale, Calif.). Glucose standards (0.1-0.0125 mg/ml diluted with 0.4% sodium hydroxide) are used to prepare a standard curve to translate the obtained A410nm values into glucose equivalents. The resultant equivalents are used to calculate the percentage of PCS cellulose conversion for each reaction.
The degree of cellulose conversion to reducing sugar (conversion, %) is calculated using the following equation:

Conversion(%)=RS(mg/ml)*100*162/(Cellulose(mg.ml)*180)==RS(mg/ml)*100/(Cellulose(mg/ml)*1.111)

In this equation, RS is the concentration of reducing sugar in solution measured in glucose equivalents (mg/ml), and the factor 1.111 reflects the weight gain in converting cellulose to glucose.

To screen for polypeptides of the invention which can enhance Celluclast® 1.5 L performance, PCS hydrolysis reactions (1.0 ml protocol, 10 g of PCS per liter, 50° C., supplemented by addition of 3% of total loading of Aspergillus oryzae beta-glucosidase) are performed in which 2.5 mg enzyme loading Celluclast® 1.5L is mixed with 2.5 mg polypeptide loading of each sample (5 mg enzyme loading Total protein per reaction). Celluclast® 1.5 L control reactions consisting of 10 mg enzyme loading, 5 mg enzyme loading and 2.5 mg enzyme loading are measured and their PCS cellulose conversion values recorded for comparison.
Example 7
Determining Lipase Activity
The culture fluid or a cell lysate of a host strain synthesising and secreting a lipase in a suitable buffer is used for measuring the activity.
Lipase assay (PNV assay): 20 microlitres of dilution buffer is pipetted into each well of a 96 well microtiter plate. 5 microlitres of sample (supernatant) is added to the dilution buffer. At the start of the assay 200 microlitres of substrate is added to each well and the plate is mounted into an ELISA reader (a programmable spectrophotometer that can read 96 well plates). Absorbance is measured at 405 nm every 30 seconds for 10 minutes. The slope of the time vs. abs405 curve is used as an arbitrary activity unit.

    Dilution buffer: 25 ml 2M Tris/HCl pH 7.5, 0.50 ml 2M CaCl2, 2.5 ml 15% Brij 35, H2O ad 500 ml.
    Substrate stock solution: 0.1295 g (117 microlitres) p-Nitrophenyl valerate SIGMA N 4377 (density 1.11 g/ml) is dissolved in 10 ml methanol. Store in freezer
    Substrate: 100 microlitres substrate stock solution is mixed with 10 ml dilution buffer.


Example 8
Determining Protease and Peptidase Activity
The culture fluid or a cell lysate of a host strain synthesising and secreting a peptidase and/or protease, in a suitable buffer having a pH chosen for optimal activity of the peptidase, may be assayed for that activity by spotting a suitable sample volume (for example 20 microliter) on an agarose plate which contain the insoluble chromogenic substrate AZCL-casein (MegazymeTM) or AZCL-collagen (MegazymeTM) OR Azocoll (Sigma-Aldrich)—e.g at a level of 0.1% w/w. The plate is incubated for an appropriate time, e.g. one day, at a temperature suitable for the function of the peptidase, e.g. 37° C. The activity is visible as blue halos around the spots. As an alternative to AZCL-casein and AZCL-collagen (MegazymeTM) non-labelled casein or non-labelled collagene can be used. On non-labelled collagen or non-labelled casein spotted on agarose plates, clearing zones form in the presence of peptidase and/or protease.
Example 9
Determining Endo-arabinase Activity
The culture fluid or a cell lysate of a host strain synthesising and secreting an arabinase in a suitable buffer having a pH chosen for optimal activity of the arabinase, may be assayed for that activity by spotting a suitable sample volume (for example 20 microliter) on an agarose plate which contain the insoluble chromogenic substrate AZCL-arabinan—e.g at a level of 0.1% w/w. The plate is incubated for an appropriate time, e.g. one day, at a temperature suitable for the function of the arabinase, e.g. 37° C. The activity is visible as blue halos around the spots.
The assay may be performed at different pH. At acidic pH the AZCL-arabinan may be prepared by dissolving 0.1 g of AZCL arabinan (Mazurine dyed and cross-linked substrate, Megazyme, Ireland) in 100 ml 0.2M Succinic acid pH3+10 microlitres TritonX-100 (0.01%), to give a final concentration of 0.1% AZCL. At neutral pH the AZCL-arabinan may be prepared by dissolving 0.1 g of AZCL arabinan (Mazurine dyed and cross-linked substrate, Megazyme, Ireland) in 50 ml sterile H2O plus 10 microlitres TritonX-100 (0.01%). 50 ml 0.4M MOPS pH 7 is then added to the 50 ml AZCL substrate, to give a final volume of 100 ml and a final concentration of 0.2M buffer, 0.1% AZCL.
Example 10
Determining Beta-glucosidase Activity
Many betaglucosidases are likely to have at least some activity on 4-nitrophenyl β-D-glucopyranoside or cellobiose even though that may not be their natural substrates. Activity may also be found using methyl-umbiliferyl beta-D-glucoside which is very sensitive. Generally any (1,4)-β- and (1,3)-β-oligoglucosides can be used as substrate to assess activity of beta-glucosidase by measuring released glucose by using the Trinder assay (The Sigma Diagnostic Glucose (Trinder) Assay, Sigma, St. Louis, Mo.).
One way of determining beta-glucosidase activity is to make a preparation of the culture fluid or a cell lysate of a host strain synthesising and secreting a beta-glucosidase so that the preparation contains approximately 6.9×10−6 mg/ml of total protein, 100 mM sodium citrate pH 5.0, 0.01% Tween-20 and 4 mM p-nitrophenyl-beta-D-glucopyranoside. The preparation is incubated at 50° C. and aliquots are taken at 0.5, 1, 2, 3, 3.75, and 24 hours. To each aliquot is added 1 M sodium carbonate pH 10.0, and the p-nitrophenyl anion concentration is determined from the absorbance at 405 nm.
Another way of determining beta-glucosidase is make a preparation of the culture fluid or a cell lysate of a host strain synthesising and secreting a beta-glucosidase by first desalting (BioRad Econo-Pac 10DG column) and then concentrating (Centricon Plus-20, Biomax-5, 5 kD cut-off), to a concentration of 0.92 mg/ml (BCA assay). Then the preparation is incubated at 0.037 and 0.0092 pg/ml total protein with 10 mM cellobiose in 100 mM sodium citrate pH 5.0 plus 0.01% Tween-20 at 65° C. Aliquots are taken at 0.5, 1, 2, 3, 4, 6, and 19 hours. Aliquots are boiled 6 minutes to terminate the reaction, and the glucose concentration is determined using the Trinder assay (Sigma Chemical Co., St. Louis, Mo.) and external glucose standards.
Example 11
Determining Esterase Activity
The culture fluid or a cell lysate of a host strain synthesising and secreting an esterase in a suitable buffer is used for measuring the activity. When choosing a substrate for detecting activity of the serine esterase one should preferably choose one which does not form micelles at the concentrations at which the esterase is saturated with substrate, to gain optimal conversion of substrate.
One way of testing esterase activity is to measure the hydrolysis of triacetin in a concentration below CMC for triacetin by the enzyme, by the alkali consumption registered as a function of time under standard conditions such as 30.0° C.; pH 7.0. Hydrolysis of triacetin by esterase will liberate acetic acid which will requires addition of alkali to maintain a a constant pH of 7.0 (pH-stat method) thus the amount of alkali (usually in the form of sodium hydroxide) required to maintain the pH at 7.0 is a measure of triacetin ester bonds hydrolysed.
Another way of testing esterase activity is to measure the hydrolysis by the esterase of PNP-acetat (para-nitrophenyl-acetate) releasing the coloured PNP. 20 microlitres of dilution buffer is pipetted into each well of a 96 well microtiter plate. 5 microlitres of sample (culture fluid supernatant or filtered cell lysate) is added to the dilution buffer. At the start of the assay 200 microlitres of substrate is added to each well and the plate is mounted into an ELISA reader (a programmable spectrophotometer that can read 96 well plates). Absorbance is measured at 405 nm every 30 seconds for 10 minutes. The slope of the time vs. abs405 curve is used as an arbitrary activity unit.

    Dilution buffer: 25 ml 2M Tris/HCl pH 7.5, 0.50 ml 2M CaCl2, 2.5 ml 15% Brij 35, H2O 13 500 ml.
    Substrate stock solution: a suitable amount of p-Nitrophenyl acetate (analytical grade) is dissolved in 10 ml methanol and stored in a freezer
    Substrate: 100 microlitres substrate stock solution is mixed with 10 ml dilution buffer.

## Claims

1. An isolated xylanase enzyme comprising an amino acid sequence which has at least 90% identity with a sequence of the mature polypeptide comprised in SEQ ID NO: 4.

2. The xylanase of claim 1, comprising an amino acid sequence which has at least 90% identity with amino acids 1-202 of SEQ ID NO: 4.

3. The xylanase of claim 1, comprising an amino acid sequence which has at least 95% identity with amino acids 1-202 of SEQ ID NO: 4.

4. The xylanase of claim 1, comprising an amino acid sequence which has at least 96% identity with amino acids 1-202 of SEQ ID NO:4.

5. The xylanase of claim 1, comprising an amino acid sequence which has at least 97% identity with amino acids 1-202 of SEQ ID NO:4.

6. The xylanase of claim 1, comprising an amino acid sequence which has at least 98% identity with amino acids 1-202 of SEQ ID NO:4.

7. The xylanase of claim 1, comprising an amino acid sequence which has at least 99% identity with amino acids 1-202 of SEQ ID NO:4.

8. The xylanase of claim 1, comprising amino acids 1-202 of SEQ ID NO:4.

9. The xylanase of claim 1, which consists of amino acids 1-202 of SEQ ID NO:4.

10. A composition comprising the xylanase of claim 1 and an excipient.

11. An isolated polynucleotide comprising a nucleotide sequence which encodes for the xylanase of claim 1.

12. A nucleic acid construct comprising the polynucleotide of claim 11 operably linked to one or more control sequences that direct the production of the xylanase in a host cell.

13. A recombinant expression vector comprising the nucleic acid construct of claim 12.

14. An isolated recombinant host cell comprising the nucleic acid construct of claim 12.

15. A method for producing a xylanase comprising:
a) cultivating the isolated recombinant host cell of claim 14 under conditions conducive for production of the xylanase; and
b) recovering the xylanase.