WO2006079346A1

WO2006079346A1 - Novel enzyme having alpha-xylosidase activity

Info

Publication number: WO2006079346A1
Application number: PCT/DK2006/000048
Authority: WO
Inventors: Christel Thea JØRGENSEN; Søren Flensted LASSEN; Allan NØRGAARD
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2005-01-28
Filing date: 2006-01-30
Publication date: 2006-08-03
Anticipated expiration: 2007-07-28

Abstract

The present invention relates to isolated polypeptides having alpha-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Description

Novel enzyme having alpha-xylosidase activity.

Field of invention

Background of the invention The patent literature is very scarce on polypeptides having alpha-xylosidase activity.

JP59048075-A disclose an acidic Aspergillus alpha-xylosidase which hydrolyses alpha bonds of xylose residues. Further, alpha-xylosidases have been mentioned as suitable for degradation of oligosaccharides (WO 02/00858, WO 00/71729).

It is an object of the present invention to provide polypeptides having alpha-xylosidase activity and polynucleotides encoding the polypeptides.

Summary of the invention

In a first aspect the present invention relates to isolated polypeptides having alpha- xylosidase activity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 99.5% identity with amino acids 1 to 769 of SEQ ID NO: 2;

(b) a polypeptide which is encoded by a nucleotide sequence which hybridizes under at least high stringency conditions with (i) nucleotides 1 to 2310 of SEQ ID NO: 1 , (ii) the cDNA sequence contained in nucleotides 1 to 2310 of SEQ ID NO: 1 , or (iii) a complementary strand of (i) or (ii); and (c) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids of amino acids 1-769 of SEQ ID NO: 2.

In a second aspect the present invention relates to isolated polynucleotides encoding polypeptides having alpha-xylosidase activity, selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acid sequence which has at least 99.5% identity with amino acids 1 to 769 of SEQ ID NO: 2;

(b) a polynucleotide having at least 99.8% identity with nucleotides 1 to 2310 of SEQ ID NO: 1 ; and (c) a polynucleotide which hybridizes under at least high stringency conditions with (i) nucleotides 1 to 2310 of SEQ ID NO: 1 , (ii) the cDNA sequence contained in nucleotides 1 to 2310 of SEQ ID NO: 1 , or (iii) a complementary strand of (i) or (ii).

The present invention also relates to nucleic acid constructs, recombinant expression vectors, and recombinant host cells comprising the polynucleotides.

The present invention also relates to methods for producing such polypeptides having alpha-xylosidase activity comprising (a) cultivating a recombinant host cell comprising a nucleic acid construct comprising a polynucleotide encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. A third aspect of the present invention relates to a method for the complete or partial removal of O-glycosylations of proteins within the epidermal growth factor-like (EGF) modules, typically on the proteins involved in the coagulation cascade in mammals, such as FVII, FVIIa, IX and IXa, thrombospondin and plasma Protein Z. The method includes contacting an O- glycosylated protein with one or more polypeptides selected from the group consisting of glycosidases such as α-xylosidases (EC 3.2.1.-), α-glucosidases (EC 3.2.1.20), glucoamylases (EC 3.2.1.3) and β-glucosidases (EC 3.2.1.21 ) and α-fucosidase (EC 3.2.1.51 ), preferably with the polypeptides of the present invention, whereby the polypeptides cleave the glycosidic bonds, such as xylosidic, fucosidic or O-glucosidic bonds to the hydroxyl group of the serine or threonine residues. A preferred embodiment of the invention is a method for producing a therapeutic protein, such as proteins involved in the coagulation cascade in mammals, including FVII, FVIIa, IX and IXa, thrombospondin and plasma Protein Z, which method comprises the steps of a) providing the protein from a host cell, and b) contacting said protein with polypeptides selected from the group consisting of glycosidases such as α-xylosidases (EC 3.2.1.-), α-glucosidases (EC 3.2.1.20), glucoamylases (EC 3.2.1.3) and β-glucosidases (EC 3.2.1 .21 ) and α-fucosidase (EC

3.2.1.51 ), preferably with the polypeptides of the present invention, whereby the said one or more polypeptides completely or partially remove the O-glycosylations of said protein.

In a fourth aspect the present invention relates to a use of a therapeutic protein for the preparation of a medicament, wherein the O-glycosylations of said therapeutic protein have been completely or partially removed.

In a fifth aspect the present invention relates to a use of an enzyme of the present invention for degradation of biomass containing xylose residues.

In a sixth aspect the present invention relates to a use of an enzyme of the present invention as an additive for a detergent composition. Definitions

Alpha-xylosidase activity: The term "alpha-xylosidase activity" is defined herein as a hydrolytic activity which catalyzes the liberation of alpha-xylose from the non-reducing terminal glucose of xyloglucan or xylan oligosaccharides. For purposes of the present invention, alpha- xylosidase activity is determined according to the procedure described in Example 3.

Isolated polypeptide: The term "isolated polypeptide" as used herein refers to a polypeptide which is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation which contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation.

The polypeptides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polypeptides are 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 by means of well-known recombinant methods or by classical purification methods.

Herein, the term "substantially pure polypeptide" is synonymous with the terms "isolated polypeptide" and "polypeptide in isolated form."

Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity". For purposes of the present invention, the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters are Ktuple=1 , gap penalty=3, windows=5, and diagonals=5. For purposes of the present invention, the degree of identity between two nucleotide sequences is determined by the Wilbur-ϋpman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters are Ktuple=3, gap penalty=3, and windows=20.

Polypeptide Fragment: The term "polypeptide fragment" is defined herein as a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of SEQ ID NO: 2 or a homologous sequence thereof, wherein the fragment has alpha- xylosidase activity. Preferably, a fragment contains at least 760 amino acid residues, more preferably at least 720 amino acid residues, more preferably 680 amino acid residues and most preferably at least 640 amino acid residues of SEQ ID NO: 2. Subsequence: The term "subsequence" is defined herein as a nucleotide sequence having one or more nucleotides deleted from the 5¹ and/or 3¹ end of SEQ ID NO: 1 or a homologous sequence thereof, wherein the subsequence encodes a polypeptide fragment having alpha-xylosidase activity. Preferably, a subsequence contains at least 2220 nucleotides, more preferably at least 2100 nucleotides, more preferably at least 1980 nucleotides and most preferably at least 1800 nucleotides.

Allelic variant: The term "allelic variant" denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Substantially pure polynucleotide: The term "substantially pure polynucleotide" as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems. Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively associated. A substantially pure polynucleotide may, however, include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polynucleotides disclosed herein are in "essentially pure form", i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively associated. Herein, the term "substantially pure polynucleotide" is synonymous with the terms "isolated polynucleotide" and "polynucleotide in isolated form." The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof. cDNA: The term "cDNA" is defined herein as a DNA molecule which can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA which is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term "nucleic acid construct" as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

Control sequence: The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.

Operably linked: The term "operably linked" denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

Coding sequence: When used herein the term "coding sequence" means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG. The coding sequence may a DNA, cDNA, or recombinant nucleotide sequence. Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term "expression vector" is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the invention, and which is operably linked to additional nucleotides that provide for its expression. Host cell: The term "host cell", as used herein, includes any cell type which is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct comprising a polynucleotide of the present invention. Modification: The term "modification" means herein any chemical modification of the polypeptide consisting of the amino acids 1 to 769 of SEQ ID NO: 2 as well as genetic manipulation of the DNA encoding that polypeptide. The modification(s) can be substitution(s), deletion(s) and/or insertions(s) of the amino acid(s) as well as replacement(s) of amino acid side chain(s). Artificial variant: When used herein, the term "artificial variant" means a polypeptide having alpha-xylosidase activity produced by an organism expressing a modified nucleotide sequence of SEQ ID NO: 1. The modified nucleotide sequence is obtained through human intervention by modification of the nucleotide sequence disclosed in SEQ ID NO: 1.

Glycoprotein: When used herein the term "glycoprotein" is intended to cover amino acids linked together by peptide bonds thereby forming a protein, which protein is glycosylated i.e. contains O-linked carbohydrate residues attached to amino acid residues serine or threonine.

Therapeutic proteins: Therapeutic proteins in the context of the present invention mean that the proteins should be administrable to a human in pure form or comprised in a pharmaceutical formulation.

Completely removed: When used herein the term "completely removed" means that in the final protein product of the method according to the invention at least 90 % is free of O- linked carbohydrate residues, particularly at least 95%, more particularly at least 97%, even more particularly at least 99%, and even more particularly 99.5%. Partly removed: When used herein the term "partly removed" means that in the final protein product of the method according to the invention at least one of the O-linked carbohydrate residues making up the O-glycosylation has been removed. In at least 90 % of the final product the at least one O-linked carbohydrate residue has been removed, particularly at least 95%, more particularly at least 97%, even more particularly at least 99%, and even more particularly in at least 99.5% of the final product, the at least one O-linked carbohydrate residue has been removed. Detailed description of the invention

Polypeptides having alpha-Xylosidase Activity

A nucleotide sequence similar to the sequence of SEQ ID NO:1 was disclosed by Rey et al. in Genome Biol. (2004) 5 (10), R77. However, Rey et al. disclosed the full genome of Bacillus licheniformis ATCC 14580 and noted a nucleotide sequence encoding a "putative family 31 glycoside hydrolase". This nucleotide sequence is 99.8% identical with SEQ ID NO:1. The sequence of the putative GH31 enzyme was published with NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) under accession no: AAU24997, but no specific activity was assigned to the polypeptide. This polypeptide sequence is 99.5% identical with SEQ ID NO:2. Family 31 glycoside hydrolases include at least seven distinct enzyme activities, the alpha-xylosidase activity being the least frequently published.

In a first aspect, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to amino acids 1 to 769 of SEQ ID NO: 2 (i.e., the mature polypeptide) of at least 99.5%, preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, even most preferably at least 99.9%, which have alpha-xylosidase activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides have an amino acid sequence which differs by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from amino acids 1 to 769 of SEQ ID NO: 2. A polypeptide of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof that has alpha-xylosidase activity. In another preferred aspect, a polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof that has alpha-xylosidase activity. In a second aspect, the present invention relates to isolated polypeptides having alpha- xylosidase activity which are encoded by polynucleotides which hybridize under medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) nucleotides 1 to 2310 of SEQ ID NO: 1 , (ii) the cDNA sequence contained in nucleotides 1 to 2310 SEQ ID NO: 1 , (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). A subsequence of SEQ ID NO: 1 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has alpha-xylosidase activity. The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having alpha-xylosidase activity 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 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Even longer probes may be used, e.g., nucleic acid probes which are at least 600 nucleotides, at least preferably at least 700 nucleotides, more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention. A genomic DNA library prepared from such other organisms may, therefore, be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having alpha-xylosidase activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA which is homologous with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labelled nucleic acid probe corresponding to the nucleotide sequence shown in SEQ ID NO: 1 , its complementary strand, or a subsequence thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using X-ray film.

In a preferred aspect, the nucleic acid probe is nucleotides 1 to 2310 of SEQ ID NO: 1. Preferably, the probe is selected from the complementary strand of 1 ) nucleotides 1 -500 of the coding DNA sequence, 2) nucleotides 800-1300 of the coding DNA sequence, or 3) nucleotides 1800-2310 of the coding DNA sequence.

In another preferred aspect, the nucleic acid probe is a polynucleotide sequence which encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof having alpha-xylosidase activity. For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS preferably at least at 45°C (very low stringency), more preferably at least at 50⁰C (low stringency), more preferably at least at 55⁰C (medium stringency), more preferably at least at 6O⁰C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70°C (very high stringency). For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post- hybridization at about 5°C to about 10⁰C below the calculated T_m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCI, 0.09 M Tris-HCI pH 7.6, 6 mM EDTA, 0.5% NP-40, 1X 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 about 70 nucleotides in length, the carrier material is washed once in 6X SCC plus 0.1 % SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5⁰C to 10°C below the calculated T_m. In a fourth aspect, the present invention relates to isolated polypeptides having the following physicochemical properties: pH optimum in the range of 5-8.5, preferably in the range of pH 6-8, most preferably at about pH 7; temperature optimum in the range of 40-65⁰C, preferably in the range of 50-60°C, most preferably at about 55°C.

In a fifth aspect, the present invention relates to artificial variants comprising a conservative substitution, deletion, and/or insertion of one or more amino acids of SEQ ID NO: 2 or the mature polypeptide thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions 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 and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter specific activity 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/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly. in addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-/V-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline. Alternatively, 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 improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e., alpha-xylosidase activity) to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. MoI. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides which are related to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. ScL USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et a/., 1991 , Biochem. 30:10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et a/., 1986, Gene 46:145; Ner et ai, 1988, DNA 7:127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. The total number of amino acid substitutions, deletions and/or insertions of amino acids

1 to 769 of SEQ ID NO: 2 is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably at most 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1.

Sources of Polypeptides Having alpha-Xylosidase Activity

A polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus polypeptide, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide; or a Streptomyces polypeptide, e.g., a Streptomyces lividans or Streptomyces murinus polypeptide; or a gram negative bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp. polypeptide.

In a preferred aspect, the polypeptide is a Bacillus licheniformis polypeptide, e.g., the polypeptide of SEQ ID NO: 2. Preferably the gene encoding the polypeptide of SEQ ID NO: 2 is obtained from the Bacillus licheniformis which is deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under number DSM 9552.

A polypeptide of the present invention may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous fungal polypeptide such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusa^ήum, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma polypeptide.

In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having alpha-xylosidase activity.

In another preferred aspect, the polypeptide is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides, Fusaήum 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, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma Iongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide may then be obtained by similarly screening a genomic or cDNA library of another microorganism. Once a polynucleotide sequence encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques which are well known to those of ordinary skill in the art (see, e.g., Sambrook et ai, 1989, supra).

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

Polynucleotides

The present invention also relates to isolated polynucleotides having a nucleotide sequence which encode a polypeptide of the present invention. In a preferred aspect, the nucleotide sequence is set forth in SEQ ID NO: 1. The present invention also encompasses nucleotide sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differs from SEQ ID NO: 1 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 1 which encode fragments of SEQ ID NO: 2 that have alpha-xylosidase activity.

The present invention also relates to mutant polynucleotides comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1, in which the mutant nucleotide sequence encodes a polypeptide which consists of amino acids 1 to 769 of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides 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., lnnis et a/., 1990, PCR- A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain of Bacillus, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides having nucleotide sequences which have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1 (i.e., nucleotides 1 to 2310) of at least 99.8%, more preferably at least 99.9% identity, and which encode a polypeptide having alpha-xylosidase activity.

Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., artificial variants that differ in specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleotide sequence presented as the polypeptide encoding region of SEQ ID NO: 1 , e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081- 1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for alpha-xylosidase activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate- enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (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).

The present invention also relates to isolated polynucleotides encoding a polypeptide of the present invention, which hybridize under medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) nucleotides 1 to 2310 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtained by (a) hybridizing a population of DNA under medium, medium-high, high, or very high stringency conditions with (i) nucleotides 1 to 2310 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); and (b) isolating the hybridizing polynucleotide, which encodes a polypeptide having alpha- xylosidase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. 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 extracellular 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, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta- glucosidase, Trichoderma reesei cellobiohydrolase I₁ Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, 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 ), Saccharo- myces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH 1 , ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et a/., 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 polypeptide. 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 ef a/., 1992, supra.

The control sequence may also be a suitable leader sequence, a nontranslated 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 polypeptide 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 polypeptide. 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 polypeptide. However, any signal peptide coding region which directs the expressed polypeptide 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 1 1837 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 polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). 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.

It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. 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.

Expression Vectors The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids 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, a 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) which can be conveniently subjected to recombinant DNA procedures and can bring about 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 which 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 which 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 which 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), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and 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 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 polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration 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 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 nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity 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. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coii, and pUB110, pE194, pTA1060, and pAMβi 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.

Examples of origins of replication useful in a filamentous fungal cell are AMA1 and

ANSI (Gems et al., 1991 , Gene 98:61-67; Cullen et a/., 1987, Nucleic Acids Research 15:

9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO

00/24883.

More than one copy of a polynucleotide 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 polynucleotide 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 polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, 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).

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide 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 term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non- unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms 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 and Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred aspect, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In another preferred aspect, 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 Thome, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell. In a preferred aspect, the host cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by

Hawksworth et a/., 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 a/., 1995, supra, page 171 ) and all mitosporic fungi (Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (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 aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

In a most preferred aspect, 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 aspect, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred aspect, 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 generally 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 aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred aspect, 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 another most preferred aspect, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogβnum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain 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 and Trichoderma 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; lto et a/., 1983, Journal of Bacteriology 153: 163; and Hinnen et a/., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. Preferably, the cell is of the genus Bacillus, and more preferably Bacillus licheniformis.

The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleotide sequence having at least one mutation in the mature polypeptide coding region of SEQ ID NO: 1 , wherein the mutant nucleotide sequence encodes a polypeptide which consists of amino acids 1 to 769 of SEQ ID NO: 2, and (b) recovering the polypeptide.

In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and 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). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein. The resulting polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The 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 sulphate precipitation), SDS-PAGE₁ or extraction (see, e.g., Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Use of the alpha-xylosidase of the invention

Q-deglvcosylation of glycoproteins

The present invention relates to polypeptides useful for modifying the O-glycans located in epidermal growth factor-like (EGF) modules, typically on the O-glycosylated proteins involved in the coagulation/fibrinolytic cascade in mammals, preferably humans. Such proteins are preferably FVII, FVIIa, IX and IXa, thrombospondin and plasma Protein Z.

The present invention further relates to a method for the modification or complete or partial removal of O-glycosylations of said proteins. The method includes contacting an O- glycosylated protein with one or more polypeptides selected from the group consisting of glycosidases such as α-xylosidases (EC 3.2.1.-), α-glucosidases (EC 3.2.1.20), glucoamylases

(EC 3.2.1.3) and β-glucosidases (EC 3.2.1.21 ) and α-fucosidase (EC 3.2.1.51), preferably the polypeptide of the present invention as shown in SEQ ID NO: 2, whereby the polypeptides cleave the glycosidic bonds, such as xylosidic, fucosidic and O-glucosidic bonds to the hydroxyl group of the serine or threonine residues.

In a preferred embodiment the present invention relates to an enzyme useful for modification of O-glycosylation of glycoproteins, wherein the enzyme exhibits alpha-xylosidase activity.

The present invention further relates to a method for producing a therapeutic protein, comprising the steps of a) providing an O-glycosylated protein from a mammal host, and b) contacting said protein with one or more of the above mentioned glycosidases, whereby the O-glycosylations of said protein are completely or partially removed. In one embodiment the O-glycosylated protein is in a folded state when contacted with the glycosidase. The term "folded" means that the protein has a secondary, and/or tertiary and/or quaternary structure during the enzymatic treatment. Further, the invention relates to the use of a therapeutic protein expressed in a mammal host for the preparation of a medicament, wherein the O-glycosylations of said therapeutic protein have been completely or partially removed by one or more of the above mentioned glycosidases, preferably by the polypeptide of the invention as shown in SEQ ID NO: 2.

Other steps or treatments that will render the O-glycosylated protein more susceptible to the glycosidase according to the invention can optionally be included. Such treatment includes e.g. denaturation by applying well known techniques, like e.g. heating, extreme pH, chemical treatment etc. Most proteins developed for pharmaceutical applications have oligosaccharides attached to specific amino acids in their polypeptide chain, when produced in a eukaryotic host cell. This is also the case for proteins produced by mammalian cells. In general sugar chains of such glycoproteins may be attached by N-glycosidic bonds to the amide group of asparagine residues or O-glycosidic bonds to the hydroxyl group of serine or threonine residues. Hereinafter the serine or threonine residues are referred to as potential sites of O- glycosylation.

The O-linked glycans do not have a common core structure, but are primarily of the mucin type, which includes 7 different core structures having either GaINAc or GIcNAc linked α-glycosidically to the hydroxyl of either serine or threonine residues (Spiro, R.G. Glycobiology 12, 43R-46R (2002)). Other types of core structures such as β-GIc and α-L-Fuc are found in epidermal growth factor (EGF) domains of multimodular proteins.

Grabenhorst et al. (Glycoconjugate Journal, 16:81-97 (1999)) reported that all mammalian cell lines frequently used for recombinant protein expression (Chinese Hamster Ovary (CHO), Baby Hamster Kidney (BHK-21), C127, Ltk^') modify O-glycosylation sites primarily with core 1 (Galβ1-→-3GalNAcα-Ser/Thr) O-glycans containing further one or two NeuAc units. Examples are recombinant haematopoietic growth factors and interferons. However, other glycan structures also appear, e.g. as seen in the coagulation factors Vll/Vlla and IX/IXa, which contain both the α-Fuc core and the β-Glc core.

Most often the glycosylation sites appear to be protein specific and not related to the host cell. Thus, lnterleukin (human IL-2) and Erythropoietin (EPO) are glycosylated at the same position in CHO and BHK-21 cells even in a cluster of potential sites. Further, FVII/FVIIa is glycosylated at the serine residues in position 52 and 60 (Ser52 and SerδO) in both plasma and recombinant FVII/FVIIa.

The effect of O-glycosylation and the specific glycoforms on the biological activity varies greatly. Bjoern et al. (J. Biol. Chem. 266, 17:11051-11057 (1991)) reported that the activity of FVIIa was not influenced by the O-glycosylation. In contrast, the O-glycosylation of erythropoietin (EPO) is of crucial importance for the activity. 0-Glycans of recombinant proteins resulting from e.g. fungal glycosylation could be recognized as antigenic structures by the human immune system and may cause unwanted side effects, such as allergic reactions or even anaphylactic shock. It is therefore desirable to modify or remove O-linked carbohydrate residues from proteins developed for pharmaceutical applications.

Modification or removal of O-glycosylations can be achieved by use of enzymes cleaving the interglycosidic bonds or the O-glycosidic bonds to the hydroxyl group of the serine or threonine residues. An endo-type of enzyme exists for part of the O-glycans. O-glycanase^® (Prozyme^®, USA), which is an endo-alpha-N-acetylgalactosaminidase is one example. This enzyme is depending on an un-substituted disaccharide core such as the core 1 structure (Galβ1→3GalNAcα-Ser/Thr) and releases unsubstituted Ser/Thr-linked GalGalNac from O- glycosylated proteins. The core 1 structure appears to be the most abundant in recombinant proteins and besides O-glycanase^®, enzymes for modification of the mucin-type O- glycosylations are available: exo-glycosidases such as neuraminidase, β-galactosidases and N-acetylglucosaminidases.

The O-glycosylations with the more unusual substitutions β-Glc-Ser/Thr and αFuc- Ser/Thr occur exclusively within the epidermal growth factor-like (EGF) modules, typically on the proteins involved in the coagulation/fibrinolytic cascade. The GIc has in all cases known been shown to be elongated with one or two xyloses. Enzymatic cleavage of the α1→3 linked xyloses has not been reported and neither has cleavage of the β-Glc-Ser bond, although it has been tried on FVIIa. Shao et al. (Glycobiology 12:763-770 (2002)) suggested that this linkage is uniquely resistant to enzymatic digestion.

It is an object of the present invention to provide enzymes capable of modifying or removing the O-glycosylations in epidermal growth factor-like (EGF) domains, typically on the proteins involved in the coagulation/fibrinolytic cascade of mammals. The enzymes of the present invention are useful for obtaining a product which is more homogeneous and standardised.

Microhomogenicity with respect to single glycoforms may in some cases be obtained by use of conventional protein purification steps. However, such procedures will increase production costs and reduce production yield.

By removing or modifying the O-glycosylations a protein product, which is more homogeneous and thus standardised to a greater extent, can be obtained. In order to avoid unwanted side effects or improve the efficiency of the protein product the greatest possible standardisation is advantageous. Further, the solubility of a protein can be altered by removal or modification of the O-glycosylation. The solubility of proteins is important e.g. when the protein is in a liquid formulation for injection or oral administration. Also, the crystallisation of the protein may be altered by modification or removal of the O-glycosylations. Such alterations may allow a higher concentration of the protein in solution. This is valuable for the manufacture of liquid formulations of the protein.

For research purposes, it may be of value to be able to remove or modify the O- glycosylations of a protein (without mutating the Ser) in order to determine the impact of these on the biological activity and immunoresponse of the protein.

The present invention relates to enzymes useful for modifying the O-glycosylations in the epidermal growth factor-like (EGF) domains of proteins typically involved in the coagulation cascade of mammals such as human and recombinant plasma coagulation factors VII, Vila, IX and IXa, thrombospondin and plasma Protein Z and recombinant mutants of these. The present invention further relates to a method for the complete or partial removal of O- glycosylations of said proteins. The method includes contacting an O-glycosylated protein with a glycosidase, such as the polypeptides of the present invention, whereby the glycosidase cleaves the glycosidic bonds, such as xylosidic, fucosidic or O-glucosidic bonds to the hydroxyl group of another sugar molecule or to the serine or threonine residues. The method can be used for the production of proteins developed for pharmaceutical applications in which cost increasing steps, such as chromatographic purification, can be avoided by using the enzymes of the invention for complete or partial removal of O-glycosylations of said proteins.

A standardisation of the O-glycosylations of the human recombinant FVII/FVIIa is of interest. FVII/FVIIa has previously been demonstrated to have O-glycosidic glycan structures at serine 52 and serine 60. Whereas serine 60 is mostly mono-fucosylated, the glycan structure at serine 52 is a disaccharide (XyI-GIc) or trisaccharide (Xyl2-Glc). The linkages are α1→3 xylosidic and β-glucosidic bonds.

For a full deglycosylation of serine 52, it is most likely that two different enzymes are needed, a glycosidase such as an α-xylosidase (EC 3.2.1.-), α-glucosidase (EC 3.2.1.20) or glucoamylase (EC 3.2.1.3) for removal of the xylose units and a β-glucosidase (EC 3.2.1.21 ) for removal of the glucose, respectively. Further, an alpha-L-fucosidase (3.2.1.51) may be needed for removal of fucoside units. For a partial processing, the α-xylosidases, α- glucosidases, alpha-fucosidases and glucoamylases may be useful for providing proteins which are homogenous in the glycosylation of serine 52 having the β-GIc present only. α-xylosidases (EC 3.2.1.-) are only reported in very few papers, and a specific EC class has not been assigned yet. However, both intra- and extra cellular enzymes are found in the literature and also a xylosidase activity hydrolyzing α1->3 linkages have been reported. α-qlucosidases (EC 3.2.1.20) are commercially available e.g. as Maltase from Genzyme Corp. USA, or from Megazyme, Ireland. Glucoamylases (EC 3.2.1.3). Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN EXTRA L and AMG™ E (from Novozymes A/S); AMIGASE™ and AMIGASE™ PLUS (from DSM); OPTIDEX™ 300, G- ZYME™ G900, G-ZYME™ and G990 ZR (from Genencor Int.). β-Glucosidases (EC 3.2.1.21) are commercially available e.g. from Megazyme, Ireland or Worthington Biochemical Corporation, US. α-L-fucosidases (3.2.1.51) are commercially available from QA-Bio, CA, USA.

Biomass degradation

The biomass material may be any material comprising cellulosic matter, for example agricultural or industrial wastes such as straw, stalks, leaves, husks, cobs, stover, rind, shells, pods, coffee fruit flesh, pineapple waste, jute waste, oil palm waste, or wood wastes such as bark, shavings, sawdust, wood pulp and pulping liquor; or farm and household waste such as manure or waste water sludge.

The predominant polysaccharide in the primary cell wall of plants and the principal constituent of biomass is cellulose; the second most abundant is hemi-cellulose. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened through polymeric lignin covalentiy cross-linked to hemicellulose. A predominant hemicellulose is xylan comprising beta-1 ,4-linked xylose residues, with alfa-1 ,2 or alfa-1 ,3 linked arabinoses. Xylan is mainly present in cell walls of monocotyledons and in wood. Another hemicellulose is xyloglucan which comprises beta-1 , 4- linked glucose, with alfa-1 ,6-linked xylose substituents. Xyloglucan is present in most plant cell walls (mainly in dicotyledons).

The polypeptide of the present invention having alpha-xylosidase activity is able to contribute to the degradation of biomass comprising xylose residues. The biomass may be degraded to fermentable sugars, which can be used for production of ethanol. An aspect of the present invention therefore relates to a method for degradation of biomass comprising contacting the biomass with a polypeptide according to SEQ ID NO: 2 or a homologous polypeptide.

Detergent applications The polypeptide of the invention may be added to and thus become a component of a detergent composition. As outlined above xylose residues are abundant in plant cell walls and thus frequently occurring in food stains comprising plant cell wall constituents. The polypeptide of the present invention having alpha-xylosidase activity is able to contribute to the degradation and removal of such food stains. The polypeptide having alpha-xylosidase activity is especially useful for removal of stains comprising components originating from fruits like cherries, raspberries and strawberries. 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 polypeptides, such as 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 Fusaήum 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 substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101 , 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

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 Lipolase™ and Lipolase

Ultra™ (Novozymes A/S).

Amylases: Suitable amylases (α and/or β) include those of bacterial or fungal origin.

Chemically modified or protein engineered mutants are included. Amylases include, for example, α-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 substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181 , 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.

Commercially available amylases are Duramyi™, Termamyl™, Fungamyl™ and BAN™

(Novozymes A/S), Rapidase™ and Purastar™ (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 US 4,435,307, US 5,648,263, US 5,691 ,178, US 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, US 5,457,046, US 5,686,593, US 5,763,254,

WO 95/24471 , WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A/S), 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 granulate, 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 US 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), polyvinyl alcohol), poly(vinylpyridine-N-oxide), poiy(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and laiiryl 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 polypeptide of the invention, having alpha-xylosidase activity, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liqour, preferably

0.05-5 mg of enzyme protein per liter of wash liqour, in particular 0.1-1 mg of enzyme protein per liter of wash liqour.

The polypeptide of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference. In conclusion an aspect of the present invention relates to a detergent composition comprising a polypeptide according to SEQ ID NO: 2.

EXAMPLE 1 : Cloning and expression of an alpha-xylosidase from Bacillus licheniformis DSM 9552. Reagents and media

Nutrient agar: Peptone 5.0 g, meat extract 3.0 g, agar 15.0 g, distilled water 1000.0 ml.

Adjust pH to 7.0, autoclave at 121⁰C, 16 minutes. Nutrient broth: Peptone 5.0 g, meat extract 3.0 g, distilled water 1000.0 ml. Adjust pH to 7.0, autoclave at 121⁰C, 16 minutes. TE: 10 mM Tris-HCI, pH 7.4, 1 mM EDTA, pH 8.0.

TEL: 50 mg/ml Lysozym in TE-buffer Thiocyanate: 5M guanidium thiocyanate, 100 mM EDTA, 0.6 % w/v N-Iaurylsarcosine, sodium salt, 60 g thiocyanate, 20 ml 0.5 M EDTA, pH 8.0, 20 ml H₂O dissolves at 65⁰C. Cool down to room temperature (RT) and add 0.6 g N- laurylsarcosine. Add H₂O to 100 ml and filter it through a 0.2 μ sterile filter. NH₄Ac: 7.5 M CH₃COONH₄.

TER: 1 μg/ml RNAse A in TE-buffer

CIA: Chloroform/isoamyl alcohol 24:1

TY*2 medium: Tryptone 40 g, yeast extract 10 g, 1 % ferrochloride 1.4 ml, 1% mangan(ll)- chloride 0.2 ml, 1 % magnesiumsulfate 3.0 ml. Add destilled water up to 1000 ml; adjust pH to 7.3 and autoclave at 121⁰C, 16 minutes.

Lysis buffer: 2OmM HEPES pH7, 2% Triton X-100, 20 μg/ml DNAse I (10 mg/ml), 1 mM

PMSF, 20 μg/ml Lysozyme (10 mg/ml).

Cloning of SEQ ID NO: 1 SEQ ID NO: 1 is the DNA sequence encoding the alpha-xylosidase from Bacillus licheniformis

DSM 9552.

Genomic DNA from Bacillus licheniformis can be isolated according to the following procedure from an over night culture in nutrient broth at 37 ⁰C:

1. Harvest 1.5 ml culture and resuspend in 100 μl TEL. Incubate at 37°C for 30 min. 2. Add 500 μl thiocynate buffer and leave at room temperature for 10 min.

3. Add 250 μl NH₄Ac and leave at ice for 10 min.

4. Add 500 μl CIA and mix.

5. Transfer to a microcentrifuge and spin for 10 min. at full speed.

6. Transfer supernatant to a new Eppendorf tube and add 0.54 volume cold isopropanol. Mix thoroughly.

7. Spin and wash the DNA pellet with 70 % EtOH.

8. Resuspend the genomic DNA in 100 μl TER.

The full DNA sequence of a Bacillus licheniformis GH31 gene was known from the Bacillus licheniformis genome (Rey et al. Genome Biology 2004, 5:R7) and can be used for design of PCR primers for amplification of the Bacillus licheniformis GH31 gene.

The genomic DNA from β. licheniformis DSM 9552 can be used as template for PCR amplification of the GH31 8. licheniformis DSM 9552 gene by standard PCR methods using primer A and primer B.

Primer A: δ'- ATGAAATTTTCAGACGGCTACTG-S' (SEQ ID NO: 3) Primer B: 5'- CGTTTTCAGCATCCTGATCACTCC-3' (SEQ ID NO: 4) The DNA coding for the GH31 gene from Bacillus licheniformis was fused by PCR to a triple promoter system (as described in WO 99/43835), consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuήngiensis crylllA promoter including stabilizing sequence and integrated by homologous recombination on the Bacillus subtilis MB1053 host cell genome (WO200395658).The gene coding for Chloramphenicol acetyl-transferase was used as maker. (Described in eg. Diderichsen.B.; Poulsen,G.B.; Joergensen.S.T,; A useful cloning vector for Bacillus subtilis. Plasmid 30:312 (1993)). PCR amplification of the expression cassette was made from some of the chloramphenicol resistant transformants to check for correct integration. Some colonies were further analyzed by DNA sequencing of the expressed gene in the cassette to confirm the correct DNA sequence, and one strain was selected.

Fermentation

The Bacillus licheniformis GH31 expressing Bacillus subtilis host cell was fermented on a rotary shaking table (250 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml TY^*2 medium supplemented with 6 μg/ml chloramphenicol, at 37⁰C for 22 hours.

Lysis of cells

Cells are harvested at 4000 rpm, 30 min. Cell pellet is resuspended in lysis buffer (a 1/10 of the culture volume) and transferred to 37 ⁰C under shaking (250 rpm) for 15 minutes. The samples are centrifuged (4000 rpm, 10 minutes) and the supematants are collected for protein purification.

EXAMPLE 2: Purification of an alpha-xylosidase from Bacillus licheniformis DSM 9552.

Buffers and solutions Anion-exchange chromatography

Loading buffer 20 mM NaH₂PO₄ pH 7.0 Elution buffer 20 mM NaH₂PO₄ + 1 M NaCI, pH 7.0

Hydrophobic interaction chromatography

Loading buffer 20 mM MES + 1 M (NH₄)₂SO₄, pH 6.0

Elution buffer 20 mM MES pH 6.0

Activity assay Substrate solution 3 mM α-D-xylopyranoside

"Stop solution" 100 mM Na₂B₄O₇ Storage

Storage buffer 20 mM NaH₂PO₄ + 0.1 mM CaCI₂, pH 7.0

All compounds were dissolved in MiIIi-Q water (resistivity 18.2 MΩ-cm).

Purification

The purification scheme consists of the following unit operations:

1. Filtration

2. Buffer exchange 3. Anion-exchange chromatography

4. Activity assay and SDS-PAGE

5. Buffer exchange

6. Hydrophobic interaction chromatography (HIC)

7. Activity assay and SDS-PAGE 8. Buffer exchange

9. Purity check

Re 1: The supernatant from the cell lysis procedure was filtered using vacuum suction and a 0.45 μm filter.

Re 2: Buffer exchange was performed by ultrafiltration in a cold room (4°C). Equipment: Amicon 8400 stirred cell from Millipore with YM10 membrane (Molecular weight cut-off: 10 kDa). The buffer was changed to anion-exchange loading buffer: 20 mM NaH₂PO₄ pH 7.0.

Re 3: Column: Q Sepharose™ Fast Flow from Amersham Biosciences. I. D. = 26 mm. Volume: 80 ml_. Flow: 10 mL/min. Gradient: 0-100% elution buffer over 15 column volumes (CV). 10 mL fractions were collected.

Re 4: In order to screen the collected fractions for activity, an alpha-xylosidase assay was performed. 25 μL sample + 50 μL substrate solution was mixed and incubated at 4O⁰C for 30 minutes. The colour formation was "boosted" by adding 150 μL "stop solution". 200 μL of the reaction mixture were transferred to a micro-titre plate and the absorbance at 405 nm measured. Based on the activity profile, fractions were selected for SDS-PAGE (4-20 % Tris-

Glycine gels, reducing conditions). The activity profile and the SDS-PAGE together formed the basis for deciding which fractions from the anion-exchange chromatography step to pool.

Re 5: Buffer exchange was performed by ultrafiltration in a cold room (4⁰C). Equipment: Amicon 8400 stirred cell from Millipore with YM10 membrane (Molecular weight cut-off: 10 kDa). The buffer was changed to HIC loading buffer: 20 mM MES + 1 M (NH₄)₂SO₄, pH 6.0. Re 6: Column: Butyl Sepharose™ 4 Fast Flow from Amersham Biosciences. I. D. = 26 mm. Volume: 80 ml_. Flow: Loading; 3 mL/min. Wash and elution; 5 mL/min. Gradient: 0-50% elution buffer over 0.5 CV. 50 % elution buffer for 1 CV. 50-100% elution buffer over 7.5 CVs. 100% elution buffer for 1.5 CVs. 10 ml_ fractions were collected.

Re 7: In order to screen the collected fractions for activity, an alpha-xylosidase assay was performed (vide supra). Based on the activity profile, fractions were selected for SDS-PAGE (4-20 % Tris-Glycine gels, reducing conditions). The activity profile and the SDS-PAGE together formed the basis for deciding which fractions from the hydrophobic interaction chromatography step to pool.

Re 8: Buffer exchange was performed by ultrafiltration in a cold room (4°C). Equipment: Amicon 8400 stirred cell from Millipore with YM 10 membrane (Molecular weight cut-off: 10 kDa). The fractions displaying alpha-xylosidase activity were pooled and the buffer was changed to 20 mM NaH₂PO₄ + 0.1 mM CaCI₂, pH 7.0. The purified product was stored in a freezer at -18°C.

Re 9: Estimation of the relative amount of full-length purified product was performed by SDS- PAGE (4-20 % Tris-Glycine gels, reducing conditions).

EXAMPLE 3, Characterisation of alpha-xylosidase from Bacillus licheniformis DSM 9552 pH-optimum

Buffers: 50 mM Britten & Robinson/0, 1 mM CaCI₂ pH 3, 4, 5, 6, 7, 8, 9 and 10 Substrate: 3 mM p-nitrophenyl-α-D-xylopyranoside (SIGMA Cat. # N1895) in MiIIi-Q water (resistivity 18.2 MΩcm).

Stop solution: 100 mM sodium tetraborate.

Control: The corresponding buffer (75 μl buffer + 50 μl substrate).

Procedure: 25 μl enzyme sample + 50 μl buffer + 50 μl substrate is mixed and incubated at 40°C in a PCR thermocycler. The colour reaction is "boosted" after 30 min. by adding 125 μl

0.1 M tetraborate. 200 μl are transferred to a micro-titre plate and the absorbance measured at

405 nm, RT.

The pH optimum is at about pH 7 as can be seen in Figure 1.

Temperature optimum

Buffer: 50 mM Britten & Robinson/0.1 mM CaCI₂ pH 7. Substrate: 3 mM p-nitrophenyl-α-d-xylopyranoside (SIGMA Cat. # N1895) in buffer solution.

Stop solution: 100 mM sodium tetraborate. Control: Buffer is used instead of enzyme solution in assay. Procedure: 25 μl enzyme sample + 50 μl buffer + 50 μl substrate (substrate is added last) are mixed and incubated at X⁰C (X is 20, 30, 40, 50, 60, 70 or 80) in a PCR thermocycler (PTC- 200 Peltier Thermal Cycler from MJ Research). The reaction is stopped after 30 min. by heating to 90⁰C for 10 minutes (heat inactivation). This is followed by cooling to 5⁰C. The colour reaction is "boosted" by adding 130 μl 0.1 M tetraborate. The reaction mixtures are transferred to 1.5 ml_ Eppendorf tubes, followed by centrifugation for 3 min. at 2O⁰C, 5000 x g. 200 μl are transferred to a micro-titre plate and the absorbance measured at 405 nm, RT.

The temperature optimum is approximately 55°C as can be seen in Figure 2.

Substrate specificity

A mixture of alpha-xylosidase (25 μL of a stock solution of ~ 0.3 mg/mL) was added to 3 mM substrate solution (50 μL, 50 mM B&R buffer, 0.1 mM CaCI₂, pH 7) and buffer (50 μL) was added. The sample was incubated at 5O⁰C. Samples are taken out and inactivated at 95⁰C for 20 min. Samples were then analysed on TLC (eluent CH₃CN/EtOAc/n-propanol/H₂O) and by HPAEC (Dionex DX-500 HPAEC-PAD system. Dionex CA, USA).

The Dionex procedure is: Dionex DX-500 HPAEC-PAD system (CarboPac PA-100 with BorateTrap columns; A buffer: 150 mM NaOH; B buffer: 150 mM NaOH + 0.6 M sodium acetate; Flow rate: 1 ml/min. Elution conditions: 0-3 min: 95% A + 5% B; 3-12 min: linear gradient: 95% A+ 5% B to 70% A and 30% B; 12-13 min: linear gradient: 70% A + 30% B to 100% B; 13-15 min: 100% B).

The alpha-xylosidase was incubated with the substrates listed in Table 1 below. It is evident from Table 1 that the enzyme has activity towards α-1 ,3-xylosidic (methyl α-1 ,3- xylobioside) and α-1 ,6-xylosidic bonds (xyloglucan oligomers). Samples taken out after 20 h showed quantitative conversion of methyl α-D-xylopyranosyl-(1->3) α-D-xylopyranoside into methyl α-D-xylopyranoside and xylose Furthermore, the alpha-xylosidase was active on pNP α-D-xylopyranoside. Methyl α-D-xylopyranoside was not hydrolyzed (product from hydrolysis of methyl α-1 ,3-xylobioside). The enzyme does not appear to hydrolyze α-D-glucopyranosidic bonds. Table t

Chemical synthesis of methyl α-D-xylopyranosyl-(1→3)-α-D-xylopyranoside (methyl α- 1 ,3-xylobioside)

¹H NMR spectra were recorded on a Varian Mercury 400 MHz at 30⁰C. Flash chromatography was accomplished using a FLASH 4Oi chromatography module from Biotage. All solvents were purchased from Merck. 2,3,4-Tri-O-benzyl-α,β-D-xylopyranose (Tejima et al, Carbohydr. Res. 7, 485-490 (1968)) and methyl 2,4-di-O-benzyl-α-D-xylopyranoside (Morishima et al, Su//. Chem. Soc. Jpn. 55, 631-632 (1982)) were prepared as described in literature.

2,3,4-Tri-O-benzyl-α,3-trichloroacetamidyl-D-xylopyranose

2,3,4-Tri-O-benzyl- α,β-D-xylopyranose (1.7 g, 4.0 mmol) was dissolved in dry CH₂CI₂ (13 ml_) and CCI₃CN (1.2 ml_, 2.9 eq.) and freshly dried K₂CO₃ (1.2 g) was added. The suspension was stirred overnight at room temperature. The mixture was then filtered and concentrated. The oil was re-dissolved in EtOAc/heptane (1 :1 ) and filtered through a short column of silica gel (2 x 5 cm) eluting with EtOAc/heptane (1 :1 ). Concentration gave 2.2 g of trichloracetimidate ready to use without further purification.

Methyl 2A2',3',4'-penta-O-benzyl-α-D-xylopyranosyl-(1-→3) g-D-xylopyranoside A mixture of 2,3,4-tri-0-benzyl-α,β-trichloroacetimidyl-D-xylopyranose (1 g, 1.7 mmol) and methyl 2,4-di-O-benzyl-α-D-xylopyranoside (0.51 g, 1.5 mmol) was dissolved in dry CH₂CI₂ (15 ml_) and cooled on a EtOH/dry ice bath. A solution of TMSOTf in CH₂CI₂ (2.24 mL, 0.033 M) was added under nitrogen. The solution was left overnight (allowed to reach room temperature) with stirring and then concentrated. The target compound was purified by flash chromatography (EtOAc/heptane 1 :3, Rf ~ 0.6) to give 183 mg of oily product. The unwanted β-anomer had a little higher Rf value. ¹H NMR (CDCI₃): 5.62 ppm (H-1 \ J = 4 Hz), 4.58 ppm (H-I ₁ J = 4 Hz). Methyl α-D-xylopyranosyl-(1->3) α-D-xylopyranoside (methyl α-1 ,3-xylobioside) Methyl 2,4,2', 3',4'-penta-O-benzyl-α-D-xylopyranosyl-(1→3) α-D-xylopyranoside (183 mg) dissolved in EtOH (3 ml_) was hydrogenated (balloon) overnight at room temperature over Pd/C (9 mg). The suspension was filtered through Celite and concentrated to give 61 mg (84%) of the title compound. ¹H NMR (CD₃OD, selected data): 5.19 ppm (H-T, J = 4 Hz), 4.66 ppm (H-1 , J = A Hz), 3.42 ppm (OCH₃). MS-MALDI-TOF: 319 (M+Na).The α-1 ,3-linkage was furthermore confirmed by one and two-dimensional ¹H NMR spectra of the peracetylated form of the title compound.

Preparation of xyloqlucan oligomers

Xyloglucan (Tamarind, 0.5 g) was dissolved in 50 imM NaOAc pH 6 (100 ml_) and the polymer incubated with EGII from A, aculeatus (50 μl_) overnight at 4O⁰C. The solution was inactivated and 95⁰C for 20 min and concentrated. The oligosaccharides were used directly without purification.

EXAMPLE 4. Modification of O-glycosylations of glycoproteins.

The O-glycosylations of glycoproteins such as those of the coagulation cascade e.g. FVII can be completely or partially removed by use of a polypeptide having alpha-xylosidase activity. The polypeptide of the invention is suitable for this purpose.

The glycoprotein is dissolved/diluted in a buffer, such as acetate, phosphate, HEPES or B&R buffer with a pH in the range of 5.0-8.5. A preferred range is pH 6.0-8.0, more preferably pH 6.5-7.5. The buffer may comprise CaCI₂, e.g. 0.01 -5mM CaCI₂ and the final concentration of the glycoprotein is 0.01-10 mg/mL, preferably 0.1-5, more preferably 0.3-3, most preferably 0.5-2 mg/mL. Alpha-xylosidase is added to a final concentration in the range of 0.00001-1.0 mg/mL, preferably 0.0001-0.8, more preferably 0.001-0.6, more preferably 0.01-0.4, most preferably 0.1-0.25 mg/mL. Protease inhibitor may be added in case there are proteolytic contaminations in the xylosidase sample. The sample is then incubated at a temperature in the interval 20-70⁰C, preferably 30-60°, more preferably 35-55⁰C until complete or partial dexylosidation has been achieved.

Deglycosylated samples can be analysed for degree of deglycosylation by mass spectroscopy and compared to the native sample, e.g. by using the procedure published by Nishimura et al. (1989) J. Biol. Chem. Vol. 264, issue 34, 20320-20325 describing the analysis of the O-glycosylation of Human Factors VII, IX, Protein Z and bovine Protein Z.

Claims

1. An isolated polypeptide having alpha-xylosidase activity, selected from the group consisting of:

(b) a polypeptide which is encoded by a polynucleotide which hybridizes under at least high stringency conditions with (i) nucleotides 1 to 2310 of SEQ ID NO: 1 , or (ii) a complementary strand of (i);

(c) a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids of amino acids 1 to 769 of SEQ ID NO: 2.

2. The polypeptide of claim 1 , which consists of SEQ ID NO: 2 or a fragment thereof having alpha-xylosidase activity.

3. The polypeptide of claim 1 , wherein the polypeptide is a variant comprising a conservative substitution, deletion, and/or insertion of one or more amino acids of amino acids 1 to 769 of SEQ ID NO: 2.

4. The polypeptide of any of claims 1-3, which has pH optimum in the range of 5-8.5 and temperature optimum in the range of 40-65⁰C.

5. An isolated polynucleotide comprising a nucleotide sequence which encodes the polypeptide of any of claims 1 -4.

6. The isolated polynucleotide of claim 5, having at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1 , in which the mutant nucleotide sequence encodes a polypeptide consisting of amino acids 1 to 796 of SEQ ID NO: 2.

7. A nucleic acid construct comprising the polynucleotide of claim 5 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

8. A recombinant expression vector comprising the nucleic acid construct of claim 7.

9. A recombinant host cell comprising the nucleic acid construct of claim 7.

10. A method for producing the polypeptide of any of claims 1-4 comprising (a) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

11. A method for producing the polypeptide of any of claims 1-4 comprising (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

12. An isolated polynucleotide obtained by (a) hybridizing a population of DNA under high stringency conditions with (i) nucleotides 1 to 2310 of SEQ ID NO: 1 , or (ii) a complementary strand of (i); and (b) isolating the hybridizing polynucleotide, which encodes a polypeptide having alpha-xylosidase activity.

13. A method for producing a polynucleotide having a mutant nucleotide sequence, comprising (a) introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO: 1, wherein the mutant nucleotide sequence encodes a polypeptide consisting of amino acids 1 to 2310 of SEQ ID NO: 2; and (b) recovering the polynucleotide comprising the mutant nucleotide sequence.

14. A mutant polynucleotide produced by the method of claim 13.

15. A method for producing a polypeptide, comprising (a) cultivating a cell comprising the mutant polynucleotide of claim 14 encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

16. A method for producing the polypeptide of any of claims 1-4, comprising (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding a polypeptide having alpha-xylosidase activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

17. A transgenic plant, plant part or plant cell, which has been transformed with a polynucleotide encoding the polypeptide of any of claims 1-4.

18. A method for the complete or partial removal of O-glycosylations of proteins within the epidermal growth factor-like (EGF) modules, comprising contacting an O-glycosylated protein with one or more polypeptides selected from the group consisting of glycosidases such as α- xylosidases (EC 3.2.1.-), α-glucosidases (EC 3.2.1.20), glucoamylases (EC 3.2.1.3) and β- glucosidases (EC 3.2.1.21 ) and α-fucosidase (EC 3.2.1.51 ).

19. The method of claim 18, wherein the O-glycosylated proteins are involved in the coagulation cascade in mammals.

20. The method of claim 19, wherein the O-glycosylated proteins are one or more of FVII, FVIIa, IX and IXa, thrombospondin and plasma Protein Z.

21. The method of claims 18-20, wherein the O-glycosylated proteins are contacted with the polypeptide according to claims 1-4.

22. A method for producing a therapeutic protein, comprising the steps of a) providing an O-glycosylated protein from a mammal host, and b) contacting said protein with one or more of the glycosidases of claim 18 or the polypeptide of claims 1-4, whereby the O-glycosylations of said protein are completely or partially removed.

23. Use of a therapeutic protein expressed in a mammal host for the preparation of a medicament, wherein the O-glycosylations of said therapeutic protein have been completely or partially removed in accordance with any of claims 18-22.

24. A detergent composition comprising a polypeptide according to any of claims 1-4.

25. A method for degradation of biomass comprising contacting the biomass with a polypeptide according to any of claims 1-4.