ZA200302197B - Talaromyces Xylanases. - Google Patents

Description

TALAROMYCES XYLANASES

.. Field of Invention

The present invention relates to a novel xylanases, such as those from Talaromyces xylanases and their use in degrading xylan in cellulose. The xylanases find use in baking, in animal feed (to improve feed conversion) and in paper production.

Background of the invention

The composition of a plant cell wall is complex and variable and contains several carbohydrate biopolymers. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising various B-xylan chains, such as xyloglucans) pectin and lignin. The most abundant hemicelluloses are xylans and their derivatives such as arabinoxylan and xyloglycan.

Plant hemicelluloses include xylan, arabinoxylan, glucuronoarabinoxylan and xyloglucan. Xylan (CAS Registry No. 9014-63-5) consists of a backbone of B-1,4-linked

D-xylopyranosyl units, optionally substituted with side chains such as arabinose and/or glucuronic acid residues. The structure is: —4)-B-D-Xylp-(1—4)-3-D-Xylp(2—1A)-( 1~4)-8-D-Xylp-(1—4)-B-D-Xylp(3<—1B)~(1— (Xylp = xylopyranosyl unit; A = a-(4-O)-methyl-(D-glucuronopyranosyl) unit, sometime an acetyl; and B = a-(L-arabinofuranosyl) unit, sometimes an acetyl).

Xylans may represent more than 30% of the dry weight of terrestrial plants. Hence . xylan is an important component of materials from natural sources that are used in industrial processes ranging from baking, improvement of animal feed conversion and paper production.

Basic differences exist between monocotyledons (e.g. cereals and grasses) and dicotyledons (e.g. clover, rapeseed and soybean) and between the seed and vegetative parts of the plant. Monocotyledons are characterized by the presence of an arabinoxylan complex as the major hemicellulose backbone, and the main structure of hemicellulose in dicotyledons is a xyloglucan complex. Higher pectin concentrations are found in

N dicotyledons than in monocotyledons. Seeds are generally high in peptic substances but relatively low in cellulosic material.

B Cellulose degrading enzymes are used for the processing of plant material in food as well as feed applications or as a food or feed additive due to of their capability to act on main plant cell wall substituents.

Most of the cellulose degrading enzymes available to the industry appear to be xylanases with a relatively low molecular weight and a moderate stability at higher temperatures. However, for certain applications it is desirable to use a xylanase with a relatively high thermostability. If a xylanase is to be used as an animal feed additive then a high thermostability is preferred because of-the high temperature conditions applied during pelleting the animal feed.

Summary of the Invention

A novel xylanase is now provided which is able to cleave B-D-xylan such as present in plant material. The xylanase may also be able to hydrolyse arabinoxylan (or have arabinoxylanase activity) and an aryl-B-D-xylopyranoside (or have xylosidase activity). . Accordingly, the present invention provides an (isolated) B-xylanase polypeptide comprising: (i) the amino acid sequence of SEQ ID No: 2; or (ii) a variant of (i) which is capable of cleaving B-D-xylan; or (iii) a fragment of (i) or (ii) which is capable of cleaving p-D-xylan.

According to another aspect of the invention there is provided a polynucleotide which comprises: . (a) the nucleic acid sequence of SEQ ID No. 1 or, a sequence encoding a polypeptide of the invention; ’ (b) a sequence which is complementary to, or which hybridises to, a sequence as defined in (a); (c) a fragment of a sequence in (a) or (b); (d) asequence having at least 60% identity to a sequence as defined in (a), (b) or (c);

or (e) asequence that is degenerate as a result of the genetic code to any of the ) sequences as defined in (a) to (d).

The invention also provides: - an (e.g. expression) vector which comprises a polynucleotide of the invention and which may be capable of expressing a polypeptide of the invention; - acell line comprising a vector of the invention; - amethod of producing a polypeptide of the invention which method comprises maintaining a cell line of the invention under conditions suitable for obtaining expression of the polypeptide and, if necessary, isolating the polypeptide; - amethod of degrading B-D-xylan, the method comprising contacting a : material comprising 3-D-xylan with a polypeptide of the invention; and - amethod for identification of a compound that modulates xylanase activity, which method comprises contacting a polypeptide of the invention with a test compound in the presence of 8-D-xylan and monitoring for or detecting any modulation of activity.

Brief Description of the Sequences

SEQ ID No. 1 is a DNA sequence encoding the xylanase of the invention from

Talaromyces emersonii;,

SEQ ID No. 2 is the amino acid sequence of the xylanase; and

SEQ ID Nos. 3 and 4 are artificial PCR primers that hybridize to SEQ ID No. 1. . Detailed Description of the Invention

A. Polynucleotides

The present invention provides an (e.g. isolated and/or purified) polynucleotide encoding a polypeptide of the invention. The present invention thus provides a polynucleotide encoding a xylanase whose amino acid sequence is set out in SEQ ID No. 2 (such as the mature sequence from amino acids 23 to 408). The present invention further ’ provides a polynucleotide encoding a polypeptide having substantial amino acid sequence ] homology to the amino acid sequence set out in SEQ ID No. 2. Also included is a polynucleotide selected from: (a) a polynucleotide comprising the nucleotide sequence (for example, from polynucleotides 69 to 1224) set out in SEQ ID No. 1, or the complement thereof; (b) a polynucleotide comprising a nucleotide sequence capable of (e.g. selectively) hybridising to a nucleotide sequence set out in SEQ ID No. 1, or a fragment thereof; (c) apolynucleotide comprising a nucleotide sequence capable of (e.g. selectively) hybridising to the complement of the nucleotide sequence set out in SEQ ID No. 1, or a fragment thereof; and/or (d) a polynucleotide comprising a polynucleotide sequence that is degenerate as a result of the genetic code to a polynucleotide defined in (a), (b) or (c).

A polynucleotide of the invention also includes a polynucleotide which: (a) encodes a polypeptide having xylanase activity, which polynucleotide is: (1) the coding sequence of SEQ ID No. 1 (for example, from polynucleotides 69 to 1224); (2) a sequence which hybridises selectively to the complement of sequence defined in (1); or (3) asequence that is degenerate as a result of the genetic code with respect to a sequence defined in (1) or (2); or (b) isa sequence complementary to a polynucleotide defined in (a).

References to SEQ. ID. No. 1 can be substituted by the mature coding sequence (polynucleotides 69 to 1224) unless the text requires otherwise. » Hybridisable sequences

The term "capable of hybridizing" means that the target polynucleotide of the invention can hybridize to the nucleic acid used as a probe (for example the nucleotide sequence set out in SEQ. ID No.1, or a fragment thereof or the complement thereof) at a level significantly above background. The invention also includes nucleotide sequences that encode for the xylanase or variants thereof as well as nucleotide sequences which are complementary thereto. The nucleotide sequence may be RNA or DNA and thus includes ’ genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence. Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the (e.g. mature) coding sequence of SEQ ID No. 1. Such nucleotides can be synthesized according to methods well known in the art’.

A polynucleotide of the invention can hybridize to the coding sequence or the complement of the (e.g. mature) coding sequence of SEQ ID No.1 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level (e.g. generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence) is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the (e.g. mature) coding sequence of SEQ

ID No. 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with *P. Selective hybridization may typically be achieved using conditions of low stringency (0.3M sodium chloride and 0.03M sodium citrate at about 40°C), medium stringency (for example, 0.3M sodium chloride and 0.03M sodium citrate at about 50°C) or high stringency (for example, 0.3M sodium chloride and 0.03M sodium citrate at about 60°C). Hybridization may be carried out under any suitable conditions known in the art! and, as a guide, low stringency can be 2 x SSC at 55°C, medium stringency can be 0.5 to 1.0 x SSC at 60°C and high stringency can be 0.1 or 0.2 x SSC at 60°C or higher (e.g. at 68°C), all at 0.5% SDS.

Modifications ; Polynucleotides of the invention may comprise DNA or RNA. They may be single or double stranded. They may also be polynucleotides which include within them one or more synthetic or modified nucleotides. A number of different types of modifications to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or addition of acridine or polylysine chains at the 3’

and/or 5” ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method ’ available in the art.

It is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism, for example in which the polypeptides of the invention are to be expressed.

The (e.g. mature) coding sequence of SEQ ID No. 1 may be modified by nucleotide substitutions, for example from or upto 1,2 or 3 to 10, 25, 50 or 100 substitutions. The polynucleotide may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends. The modified polynucleotide generally encodes a polypeptide which has xylanase activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as discussed with reference to polypeptides later.

Homologues :

A nucleotide sequence which is capable of selectively hybridizing to (e.g. the complement of) the DNA coding sequence of SEQ ID No. 1 (or nucleotides 69-1224) may have at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity (or homology) to the coding sequence of SEQ ID No. 1. This may be over a region of at least 20, preferably at least 30 or 60, for instance at least 100, at least 200, more preferably at least 300 contiguous nucleotides or optimally over the full length of SEQ ID No. 1.

Any combination of the above mentioned degrees of homology and minimum sized may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher homology over longer lengths) being preferred. Thus for : example a polynucleotide which is at least 80% or 90% homologous over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which is at least 90% homologous over 40 nucleotides.

Homologues of polynucleotide (or protein) sequences typically have at least 70% homology, preferably at least 80, 90%. 95%, 97% or 99% homology, for example over a region of at least 20, 25, 30, 100 more contiguous nucleotides (or amino acids). The homology may calculated on the basis of amino acid identity (sometimes referred to as ’ "hard homology").

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings’). The PILEUP and

BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences, for example on their default settings®’).

Software for performing BLAST analyses is publicly available through the National

Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold’. These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

The BLAST program uses as defaults a word length (W) of 11, the BLOSUMS62 scoring matrix® alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences’. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match . between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in ) comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Primers and Probes ’ Polynucleotides of the invention include and may be used as a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least or up to 20, 25, 30 or 40, for example at least 25, 30 or 40 nucleotides in length. They will typically be up to 30, 40, 50, 60, 70, 100, 150, 200 or 300 nucleotides in length, or this number (even up to as few nucleotides as 5 or 10 nucleotides) short of the (e.g. mature) coding sequence of SEQ ID No. 1.

In general, primers will be produced by synthetic means, involving a step-wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Examples of primers of the invention are set out in SEQ ID Nos 3 and 4.

Longer polynucleotides will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the xylanase which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from a target (e.g. yeast, bacterial, plant, prokaryotic or fungal) cell, preferably of an Talaromyces strain, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

Such techniques may be used to obtain all or part of the xylanase sequence described herein. Genomic clones corresponding to the cDNA of SEQ ID No. 1 or the

Xylanase gene containing, for example, introns and promoter regions are within the : invention also and may also be obtained in an analogous manner (e.g. recombinant means,

PCR, cloning techniques), starting with genomic DNA from a fungal, yeast, bacterial plant or prokaryotic cell.

The polynucleotides or primers may carry a revealing label, e.g. a radioactive or non- radioactive label. Suitable labels include radioisotopes such as *P or *S, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers of the invention and may be detected using techniques known per se.

Polynucleotides, labelled or unlabelled may be used in nucleic acid-based tests for ’ detecting or sequencing Xylanase or a variant thereof in a (e.g. fungal) sample. Such tests for detecting generally comprise bringing a (e.g. fungal) sample (suspected of) containing

DNA into contact with a probe or primer of the invention under hybridizing conditions and detecting any duplex formed between the probe and nucleic acid in the sample. Such detection may be achieved using techniques such as PCR or by immobilizing the probe on a solid support, removing nucleic acid in the sample which is not hybridized to the probe, and then detecting nucleic acid which was hybridized to the probe. Alternatively, the sample nucleic acid may be immobilized on a solid support, and the amount of probe bound to such a support can be detected.

The probes of the invention may conveniently be packaged in the form of a test kit in a suitable container. In such kits the probe may be bound to a solid support where the assay format for which the kit is designed requires such binding. The kit may also contain suitable reagents for treating the sample to be probed, hybridizing the probe to nucleic acid in the sample, control reagents, instructions, and the like.

Preferably, the polynucleotide of the invention is obtainable from the same organism as the polypeptide, such as a fungus, in particular a fungus of the genus Talaromyces.

The polynucleotides of the invention also include variants of the sequence of SEQ ID

No. 1 which have xylanase activity. Variants may be formed by additions, substitutions and/or deletions and may have the ability to cleave a B-D-xylan polymer.

Production of polynucleotides

Polynucleotides which do not have 100% identity with (e.g. the mature coding sequence of) SEQ ID No. 1 but fall within the scope of the invention can be obtained in a number of ways. Thus variants of the xylanase sequence described herein may be obtained for example by probing genomic DNA libraries made from a range of organisms, for ) example those discussed as sources of the polypeptides of the invention. In addition, other fungal, plant or prokaryotic homologues of xylanase may be obtained and such homologues and fragments thereof in general will be capable of hybridising to SEQ ID No. 1. Such sequences may be obtained by probing cDNA libraries or genomic DNA libraries from other species, and probing such libraries with probes comprising all or part of SEQ

ID. 1 under conditions of medium to high stringency (as described earlier). Nucleic acid ’ probes comprising all or part of SEQ ID No. 1 may be used to probe cDNA libraries from other species, such as those described as sources for the polypeptides of the invention.

Species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences. The primers can contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of the xylanase sequences or variants thereof. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

The invention includes double stranded polynucleotides comprising a polynucleotide of the invention and its complement.

The present invention also provides polynucleotides encoding the polypeptides of the invention described below. Since such polynucleotides will be useful as sequences for recombinant production of polypeptides of the invention, it is not necessary for them to be capable of hybridising to the sequence of SEQ ID No. 1, although this will generally be desirable. Otherwise, such polynucleotides may be labelled, used, and made as described above if desired. DNA fragments may be prepared by using the PCR technique with specific primers.”**

B. Polypeptides.

The present invention relates to an (e.g. (substantially) purified and/or isolated) xylanase and variants thereof. The polypeptides of the invention may consist essentially of the amino acid sequence of SEQ ID No. 2, or a part of it (such as the mature sequence from positions 23 to 408), or a variant thereof. Polypeptides may also be encoded by a polynucleotide of the invention as described above. References to SEQ. ID. No. 2 can be substituted with the mature sequence only (residues Ala” to Leu) unless the context requires otherwise. ’ The polypeptides of the invention can be active on both arabinoxylan and aryl-p-D-xylosides (such as have arabinoxylanase and xylosidase activity).

A polypeptide of the invention may be in an isolated or a substantially purified form.

It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose and/of function of the polypeptide and still be regarded as substantially isolated. It will generally comprise the polypeptide in a preparation in which more than 20%, e.g. more than 30%, 40%, 50%, 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.

These are relatively pure compositions: for some applications the polypeptide may be present in the composition at up to 10%, 5%, 2%, 1% or even no more than 0.5%. Routine methods can be employed to purify and/or synthesise the proteins according to the invention'. For some formulations (e.g. for non-pharmaceutical uses) the amount of polypeptide present may be small, for example from 0.01 to 10%, such as from 0.1 to 5%, or 2% or even from 0.2 to 1%.

Preferably, the polypeptide of the invention is obtainable from a microorganism which. possesses a gene encoding an enzyme with xylanase activity. More preferably the microorganism is a fungus, and optimally a filamentous fungi. Preferred organisms are thus of the genus Talaromyces, such as of the species Talaromyces emersonii (e.g. CBS 393.64 or 814.70).

Activity

A polypeptide of the invention can have one or more of the following features, namely it: . (1) possesses p-D-xylanase activity; (2) has an optimum pH range of from 2 to 6, such as from 3 to 5, optimally from 3.5 ’ to 5.0; (3) has optimum activity at a temperature of from 50°C to 95°C, such as 70 to 90°C, optimally from 75 to 85°C; (4) has a molecular weight (deglycosylated) of from 30 to 50 kDa, preferably from to 45 kDa, optimally from 40 to 44 kDa or (glycosylated) of from 50 to 75kDa, preferably from 55 to 70kDa, optimally from 60 to 66kDa; and/or ) (5) has an isoelectric point of from 3.0 to 3.6.

The polypeptide can have the activity of EC.3.2.1.8. Preferably the polypeptide is from Family 10 (formerly F-type). "Xylanase activity" is defined as the ability to cleave cellulose or a B-D-xylan polymer : (for example as found in plants e.g. oat or barley). The activity thus allows cleavage of p-D-xylan, such as between adjacent xylopyranosyl terminal and/or non-terminal units.

Preferably the cleavage occurs at a [xylopyranosyl (1-4) xylopyranosyl] linkage. The polypeptide may preferentially cleave in between two adjacent (e.g. non-substituted) units.

It can thus have endo activity (i.e. be an endoxylanase). The substrate polymer may or may not be substituted. It may also have exo activity (i.e. be an exoxylanase), such as cleavage of terminal xylopyranosyl units. Preferably the polypeptide will not have glucanase activity.

Polypeptides of the invention may also be active (or display activity) on arabinoxylan.

Arabinoxylan is a sub-set of xylan, with L-arabino-furanosyl side chains linked to the C-2 - or C-3, or both, of the xylos main chain residues. Arabinoxylan has the CAS Registry No. 98513-12-3. It can have the structure (1—>4)-B-D-xylan with 3-linked o-L-arabinose branches. This type of xylan is normally found in oat spelt xylan.

This activity is the ability to hydrolyse untreated arabinoxylan. This means that the arabinoxylan has not been treated or modified, for example it has not been treated with an arabinofuranosidase. This enzyme can remove arabinose side chains. The polypeptides of the invention are able to hydrolyse (cleave) arabinoxylan that has not been prior treated with arabinofuranosidase.

Arabinoxylan can be found in oats spelts, and in this specification the activity of the polypeptide (EXU, as well as PAHBAH activity) is determined on arabinoxylan from wheat flour (with an arabinose: xylose ratio of 41:59). An assay for arabinoxylan (as the substrate) is described later in the Examples.

The polypeptides of the invention may also have xylosidase activity, for example be able to hydrolyse substituted (e.g. aryl)-p-D-xylosides (also known as xylopyranosides).

For example, they may be able to hydrolyse 4-methylumbelliferyl-B-D-xylopyranoside (CAS Registry No. 6734-33-4, obtainable from Sigma Chemical Co). This activity is the ability to liberate the fluorescent marker form the substrate. It may also hydrolyse (be active on) 5-bromo-4-chloro-3-indoxyl-R-D-xylopyranoside (CAS Registry No. 207606- ‘ 55-1). The combination of activity on both arabinoxylan and an aryl-p-D-xyloside is unusual®**’ and is a novel combination of activities for a polypeptide having xylanase activity.

Variants and Homologues

A polypeptide of the invention can comprise the amino acid sequence set out in SEQ

ID No. 2 or a substantially homologous sequence, or a fragment of either sequence and can have xylanase activity. In general, the naturally occurring amino acid sequence shown in

SEQ ID No. 2 is preferred.

In particular, the polypeptide of the invention may comprise: a. the (mature) polypeptide sequence of SEQ ID No. 2 (residues 23 to 408) or the entire sequence of SEQ ID No. 2; b. a naturally occurring variant or species homologue thereof; or c. aprotein with at least 70, at least 75, at least 80, at least 90, at least 95, at least 98 or at least 99% sequence identity to (a) or (b).

A variant may be one that occurs naturally, for example in fungal, bacteria, yeast or . plant cells and which can function in a substantially similar manner to the protein of

SEQ ID No. 2, for example it has xylanase activity. Similarly a species homologue of the protein will be the equivalent protein which occurs naturally in another species and which can function as a xylanase. Variants include allelic variants either from the same strain as the polypeptide of the invention or from a different strain, but of the same genus, or of the same species.

Variants and species homologues can be obtained by following the procedures : described herein for the production of the polypeptide of SEQ ID No. 2 and performing such procedures on a suitable cell source, for example a bacterial, yeast, fungal or plant cell. It will also be possible to use a probe as defined above to probe libraries made from yeast, bacterial, fungal or plant cells in order to obtain clones including the variants or species homology. The clones can be manipulated by conventional techniques to generate a polypeptide of the invention which can then be produced by recombinant or synthetic techniques known per se.

The polypeptide of the invention preferably has at least 70% sequence identity to the protein of SEQ ID No. 2, more preferably at least 80%, at least 90%, at least 95%, at east 97% or at least 99% sequence identity thereto, for example over a region of at least 40, 60, : 100, 150, 200, 300 or 400 contiguous amino acids or over the full length of SEQ ID No. 2.

The sequence of the polypeptide of SEQ ID No. 2 and of variants and species homologues can thus be modified to provide polypeptides of the invention. Amino acid substitutions may be made, for example from or up to 1, 2 or 3 to 10, 20, 30, 50 or 100 substitutions. The same number of deletions or insertions may also be made. These changes may be made outside regions critical to the function of the polypeptide and so may still result in an active enzyme. The modified polypeptide generally retains activity as a

Xylanase.

Polypeptides of the invention include fragments of the above mentioned full length polypeptides and of variants thereof, including fragments of the sequence set out in SEQ

ID No. 2. Such fragments typically retain activity as a xylanase. Fragments may be at least 50, 60, 70, 80, 100, 150, 200 or 250 amino acids long or may be this number of amino acids short of the full length sequence (shown in SEQ ID No. 2). Fragments or variants comprise or represent a 3-D-xylan binding region or a 8-D-xylan cleaving region.

Polypeptides of the invention can if necessary be produced by synthetic means although usually they will be made recombinantly as described below. They may be modified for example by the addition of histidine residues or a T7 tag to assist their identification or purification or by the addition of a signal sequence to promote their secretion from a cell.

The term "variants" refers to polypeptides which can have the same essential character or basic biological functionality as the xylanase, and include allelic variants. The essential character of xylanase is that it is an enzyme that can cleave 1-4 links in B-D-xylan. A ‘ polypeptide having the same essential character as the xylanase may be identified by using a cellulose degradation assay as described later.

Variants of SEQ ID No.2 also include sequences which vary from SEQ ID No.2 but which are not necessarily derived from the naturally occurring xylanase protein. These variants may be described as having a % homology to SEQ ID No.2 or having a number of substitutions within this sequence. Alternatively a variant may be encoded by a polynucleotide which hybridizes to SEQ ID No 1.

The variants can be defined in a similar manner to the variants of SEQ ID No. 1. Thus the variants may comprise variant sequences derived from other strains of Talaromyces.

Other variants can be identified from other Talaromyces strains by looking for xylanase activity and cloning and sequencing as before. Variants may include the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains the basic biological functionality of the xylanase.

Conservative substitutions may be made, for example according to the following

Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. Preferably substitutions do not affect the folding or activity of the polypeptide.

Polar-uncharged

Polar-charged

AROWATC | [HRWY

Modifications

Polypeptides of the invention may be chemically modified, e.g. post-translationally modified. For example, they may be glycosylated (one or more times, by the same or different sugars) or comprise modified amino acid residues. They may also be modified by the addition of histidine residues (to assist their purification) or by the addition of a signal ) sequence (to promote insertion into the cell membrane). The polypeptide may have one or more (N) amino- or (C) carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a (small) extension that facilitates purification, such as a poly-histidine or T7 tag, an antigenic epitope or a (e.g. maltose) binding domain" (e.g. at the C-terminus). These extensions may or may not be added via a linker.

A polypeptide of the invention may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. '*’I, *’S, enzymes, antibodies, polynucleotides and linkers such as biotin.

The polypeptides may be modified to include non-naturally occurring amino acids or to increase the stability of the polypeptide. When the proteins or peptides are produced by synthetic means, such amino acids may be introduced during production. The proteins or peptides may also be modified following either synthetic or recombinant production.

The polypeptides of the invention may also be produced using, or comprise (one or more) D-amino acids. In such cases the amino acid residues can be linked using the conventional N to C sequence as described in this application.

A number of side chain modifications are known in the art and may be made to the side chains of the proteins or peptides of the present invention. Such modifications include, for example, modifications of amino acids by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH,, amidination with methylacetimidate or acylation with acetic anhydride.

The sequences provided by the present invention may also be used as starting materials for the construction of "second generation" enzymes. "Second generation” : xylanases can be those that have been altered by mutagenesis techniques (e.g. site-directed mutagenesis), which have properties that differ from those of wild-type xylanases or recombinant xylanases such as those produced by the present invention. For example, the temperature or pH optimum, specific activity, substrate affinity or thermostability may be altered so as to be better suited for application in a defined process.

Amino acids essential to the activity of the xylanases of the invention, and therefore preferably subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis'®. In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. xylanase activity) to identify amino ’ acid residues that are critical to the activity of the molecule. Sites of enzyme-substrate interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photo-affinity labelling!!1>!® or molecular modelling.

The use of yeast and fungal host cells is expected to provide for such post-translational modifications (e.g. proteolytic processing, myristilation, glycosylation, truncation, and ' tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.

Polypeptides of the invention may be provided in a form such that they are outside their natural cellular environment. Thus, they may be substantially isolated or purified, as discussed above, or in a cell in which they do not occur in nature, e.g. a cell of other fungal species, animals, yeast or bacteria.

C. Recombinant Aspects.

The invention also provides vectors comprising a polynucleotide of the invention, including cloning and expression vectors, and methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a polypeptide of the invention occurs. Provided also are host cells comprising a polynucleotide or vector of the invention wherein the polynucleotide is heterologous to the genome of the host cell. The term "heterologous", usually with respect to the host cell, means that the polynucleotide does not naturally occur in the genome of the host cell or that the polypeptide is not naturally produced by that cell. Preferably, the host cell is a yeast cell, for example a yeast cell of the genus Kluyveromyces or

Saccharomyces or a fungal cell, for example of the genus Aspergillus.

Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a o compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.

Vectors ’ The polynucleotide of the invention may inserted into an expression cassette. The vector into which the expression cassette or polynucleotide of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced.

Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

Preferably, a polynucleotide of the invention in a vector is operably linked to a regulatory sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences or the sequences are arranged so that they function in concert for their intended purpose, for example transcription initiates at a promoter and proceeds through the DNA sequence encoding the polypeptide.

The vector may be a plasmid, cosmic, virus or phage vector, usually provided with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally an enhancer and/or a regulator of the promoter. A terminator sequence may be present, as may be a polyadenylation sequence. The vector may contain one or more selectable marker genes, for example an ampicillin resistance gene (in the case of a : bacterial plasmid) or a neomycin resistance gene (for a mammalian vector). Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. They may comprise two or more polynucleotides of the invention, for example for overexpression.

The DNA sequence encoding the polypeptide is preferably introduced into a suitable host as part of an expression cassette (or construct) in which the DNA sequence is operably linked to expression signals which are capable of directing expression of the DNA sequence in the host cells. For transformation of the suitable host with the expression construct transformation procedures are available which are well known to the skilled person™*. The expression construct can be used for transformation of the host as part of a vector carrying a selectable marker, or the expression construct may be co-transformed as a separate molecule together with the vector carrying a selectable marker. The vector may comprise one or more selectable marker genes.

Preferred selectable markers'™'® include but are not limited to those that complement a defect in the host cell or confer resistance to a drug. They include e.g. versatile marker genes that can be used for transformation of most filamentous fungi and yeasts such as acetamidase genes or cDNAs (the amdsS, niaD, facA genes or cDNAs from A. nidulans,

A.oryzae, or A.niger), or genes providing resistance to antibiotics like G418, hygromycin, bleomycin, kanamycin, phleomycin or benomyl resistance (benA). Alternatively, specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e.g. URA43 (from S.cerevisiae or analogous genes from other yeasts), pyrG or pyrd (from A.nidulans or A.niger), argB (from A.nidulans or A.niger) or tipC. Ina preferred embodiment the selection marker is deleted from the transformed host cell after introduction of the expression construct so as to obtain transformed host cells capable of producing the polypeptide which are free of selection marker genes®*.

Other markers include ATP synthetase, subunit 9 (0/iC), orotidine-5’-phosphate- decarboxylase (pvrA), the bacterial G418 resistance gene (this may also be used in yeast, but not in fungi), the ampicillin resistance gene (E. coli), the neomycin resistance gene (Bacillus) and the E. coli uidA gene, coding for B-glucuronidase (GUS). Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

For most filamentous fungi and yeast, the vector or expression construct is preferably . integrated in the genome of the host cell in order to obtain stable transformants. However, for certain yeasts also suitable episomal vectors are available into which the expression construct can be incorporated for stable and high level expression, examples thereof include vectors derived from the 2p and pKD1 plasmids of Saccharomyces and

Kluyveromyces, respectively, or vectors containing an AMA sequence (e.g. AMAI1 from

Aspergillus®*®). In case the expression constructs are integrated in the host cells genome,

the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a ) highly expressed gene. A highly expressed gene is a gene whose mRNA can make up at least 0.01% (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 0.2% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.05g/1. A number of examples of suitable highly expressed genes are provided below.

A vector or expression construct for a given host cell may comprise the following elements operably linked to each other in a consecutive order from the 5'-end to 3'-end relative to the coding strand of the sequence encoding the polypeptide of the first invention: (1) a promoter sequence capable of directing transcription of the DNA sequence encoding the polypeptide in the given host cell; (2) optionally, a signal sequence capable of directing secretion of the polypeptide from the given host cell into a culture medium; (3) a DNA sequence encoding a mature and preferably active form of the polypeptide; and preferably also (4) a transcription termination region (terminator) capable of terminating transcription downstream of the DNA sequence encoding the polypeptide.

Downstream of the DNA sequence encoding the polypeptide there may be a 3' untranslated region containing one or more transcription termination sites (e.g. a terminator). The origin of the terminator is less critical. The terminator can e.g. be native to the DNA sequence encoding the polypeptide. However, preferably a yeast terminator is used in yeast host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell (in which the

DNA sequence encoding the polypeptide is to be expressed). : Enhanced expression of the polynucleotide encoding the polypeptide of the invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and/or terminator regions, which may serve to increase expression and, if desired, secretion levels of the protein of interest from the expression host and/or to provide for the inducible control of the expression of the polypeptide of the invention.

Aside from the promoter native to the gene encoding the polypeptide of the invention,

other promoters may be used to direct expression of the polypeptide of the invention. The promoter may be selected for its efficiency in directing the expression of the polypeptide of : the invention in the desired expression host.

Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. For example prokaryotic promoters may be used, in particular those suitable for use in E.coli strains.

When expression is carried out in mammalian cells, mammalian promoters may be used.

Tissues-specific promoters, for example hepatocyte cell-specific promoters, may also be used. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), promoter rouse sarcoma virus (RSV) LTR promoter,

SV40 (e.g. large T antigen) promoter, human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters or adenovirus promoters, HSV promoters such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR).

Yeast promoters include S. cerevisiae GAL4 and ADH promoters, the S. pombe nmt 1 and adh promoter. Mammalian promoters include the metallothionein promoter which may be induced in response to heavy metals such as cadmium and 8-actin promoters. Tissue- specific promoters, in particular endothelial or neuronal cell specific promoters (for example the DDAHI and DDAHII promoters), are especially preferred.

A variety of promoters'>'® can be used that are capable of directing transcription in the host cells of the invention. Preferably the promoter sequence is derived from a highly expressed gene as previously defined. Examples of preferred highly expressed genes from which promoters are preferably derived and/or which are comprised in preferred predetermined target loci for integration of expression constructs, include but are not limited to genes encoding glycolytic enzymes such as triose-phosphate isomerases (TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate kinases (PGK), pyruvate kinases (PYK or PKI), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamylases, proteases, xylanases, cellobiohydrolases, 8-galactosidases, alcohol (methanol) oxidases, elongation factors and ribosomal proteins. Specific examples ‘ of suitable highly expressed genes include e.g. the LAC4 gene from Kluyveromyces sp., the methanol oxidase genes (40X and MOX) from Hansenula and Pichia, respectively, the glucoamylase (glad) genes from A.niger and A.awamori, the A.oryzae TAK A-amylase gene, the A.nidulans gpdA gene and the T reesei cellobiohydrolase genes.

Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts'*'*** are those which are obtainable from the fungal genes for xylanase (x/nA), phytase, ATP-synthetase, subunit 9 (0/iC), triose phosphate isomerase (tpi), alcohol dehydrogenase (4dhA), e-amylase (amy), amyloglucosidase (AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) promoters.

Examples of strong yeast promoters are those obtainable from the genes for alcohol dehydro genase, lactase, 3-phosphoglycerate kinase and triosephosphate isomerase.

Examples of strong bacterial promoters are the a-amylase and SPo2 promoters as well as promoters from extracellular protease genes.

The native promoter of the gene encoding a xylanase may be replaced by a promoter that is regulated differently than the native promoter.

Promoters suitable for plant cells include napaline synthase (nos), octopine synthase (ocs), mannopine synthase (mas), ribulose small subunit (rubisco ssu), histone, rice actin, phaseolin, cauliflower mosaic virus (CMV) 35S and 19S and circovirus promoters. All these promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to

RNA which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors!" and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses (such as HPV-16 or

HPV-18). Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the antisense RNA into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

The vector may contain a polynucleotide of the invention oriented in an antisense direction to provide for the production of antisense RNA. This may be used to reduce, if desirable, the levels of expression of the polypeptide.

Host cells and Expression

In a further aspect the invention provides a process for preparing a polypeptide according to the invention which comprises cultivating a host cell (e.g. transformed or transfected with an expression vector as described above) under conditions to provide for expression (by the vector) of a coding sequence encoding the polypeptide, and optionally recovering the expressed polypeptide. Polynucleotides of the invention can be incorporated into a recombinant replicable vector, e.g. an expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about the replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect cells such as S9 cells and (e.g. filamentous) fungal cells.

Preferably the polypeptide is produced as a secreted protein in which case the DNA sequence encoding a mature form of the polypeptide in the expression construct is operably linked to a DNA sequence encoding a signal sequence. Preferably the signal sequence is native (homologous) to the DNA sequence encoding the polypeptide. Alternatively the signal sequence is foreign (heterologous) to the DNA sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the

DNA sequence is expressed. Examples of suitable signal sequences for yeast host cells are the signal sequences derived from yeast o-factor genes. Similarly, a suitable signal sequence for filamentous fungal host cells is e.g. a signal sequence derived from a filamentous fungal amyloglucosidase (AG) gene, e.g. the 4.niger glad gene. This may be : used in combination with the amyloglucosidase (also called (gluco)amylase) promoter itself, as well as in combination with other promoters. Hybrid signal sequences may also be used with the context of the present invention.

Preferred heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from

Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces and Kluyveromyces) or the a-amylase gene (Bacillus).

The vectors may be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptide.

A further aspect of the invention thus provides host cells transformed or transfected with or comprising a polynucleotide or vector of the invention. Preferably the polynucleotide is carried in a vector for the replication and expression of the polynucleotide. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.

A heterologous host may also be chosen wherein the polypeptide of the invention is produced in a form which is substantially free from other cellulose-degrading enzymes.

This may be achieved by choosing a host which does not normally produce such enzymes such as Kluyveromyces lactis.

The invention encompasses processes for the production of the polypeptide of the invention by means of recombinant expression of a DNA sequence encoding the polypeptide. For this purpose the DNA sequence of the invention can be used for gene amplification and/or exchange of expression signals, such as promoters, secretion signal sequences, in order to allow economic production of the polypeptide in a suitable homologous or heterologous host cell. A homologous host cell is a host cell which is of the same species or which is a variant within the same species as the species from which the DNA sequence is derived.

Suitable host cells are preferably prokaryotic microorganisms such as bacteria, or more preferably eukaryotic organisms, for example fungi, such as yeasts or filamentous fungi, or plant cells. In general, yeast cells are preferred over fungal cells because they are , easier to manipulate. However, some proteins are either poorly secreted from yeasts, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these ) instances, a fungal host organism should be selected.

The host cell may over-express the polypeptide, and techniques for engineering over-expression are well known’. The host may thus have two or more copies of the encoding polynucleotide (and the vector may thus have two or more copies accordingly).

Bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts : are those from the genera Streptomyces and Pseudomonas. A preferred yeast host cell for the expression of the DNA sequence encoding the polypeptide is of the genera ’ Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, and Schizosaccharomyces.

More preferably a yeast host cell is selected from the group consisting of the species

Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var. lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica,and

Schizosaccharomyces pombe.

Most preferred are, however, (e.g. filamentous) fungal host cells. Preferred : filamentous fungal host cells are selected from the group consisting of the genera

Aspergillus, Trichoderma, Fusarium, Disporotrichum, Penicillium, Acremonium,

Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia, and Talaromyces.

More preferably a filamentous fungal host cell is of the species Aspergillus oyzae,

Aspergillus sojae, Aspergillus nidulans, or a species from the Aspergillus niger Group”.

These include but are not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubingensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans, Aspergillus

Japonicus, Aspergillus oryzae and Aspergillus ficuum, and further consisting of the species

Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium alabamense, Neurospora crassa, Myceliophtora thermophilum, Sporotrichum cellulophilum, Disporotrichum dimorphosporum and Thielavia terrestris.

Examples of preferred expression hosts within the scope of the present invention are fungi such as Aspergillus species’? and Trichoderma species; bacteria such as Bacillus species™?, e.g. Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens,

Pseudomonas species; and yeasts such as Kluyveromyces species”, e.g. Kluyveronmyces lactis” and Saccharomyces species, e.g. Saccharomyces cerevisiae. . Host cells according to the invention include plant cells, and the invention therefore extends to transgenic organisms, such as plants and parts thereof, which contain one or : more cells of the invention. The cells may heterologously express the polypeptide of the invention or may heterologously contain one or more of the polynucleotides of the invention. The transgenic (or genetically modified) plant may therefore have inserted (e.g. stably) into its genome a sequence encoding one or more of the polypeptides of the invention. The transformation of plant cells can be performed using known techniques, for example using a Ti or a Ri plasmid from Agrobacterium tumefaciens. The plasmid (or : vector) may thus contain sequences necessary to infect a plant, and derivatives of the Ti and/or Ri plasmids may be employed.

Alternatively direct infection of a part of a plant, such as a leaf, root or stem can be effected. In this technique the plant to be infected can be wounded, for example by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then innoculated with the Agrobacterium. The plant or plant part can then be grown on a suitable culture medium and allowed to develop into a mature plant. Regeneration of transformed cells into genetically modified plants can be achieved by using known techniques, for example by selecting transformed shoots using an antibiotic and by sub-culturing the shoots on a medium containing the appropriate nutrients, plant hormones and the like."

Culture of host cells and recombinant production

The invention also includes cells that have been modified to express the xylanase or a variant thereof. Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast and (e.g. filamentous) fungal cells or prokaryotic cells such as bacterial cells.

It is also possible for the proteins of the invention to be transiently expressed in a cell line or on a membrane, such as for example in a baculovirus expression system. Such systems, which are adapted to express the proteins according to the invention, are also included within the scope of the present invention.

According to the present invention, the production of the polypeptide of the invention can be effected by the culturing of microbial expression hosts, which have been . | transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium. ’ The recombinant host cells according to the invention may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture condition are available which are conducive to the expression the DNA sequence encoding the polypeptide. After reaching the desired cell density or titre of the polypeptide the

Claims (33)

1. A xylanase polypeptide comprising: 6) the amino acid sequence from amino acids 23 to 408 of SEQ ID No. 2; or (ii) a variant of (i) which is capable of cleaving xylan wherein the variant (ii) has at least 70% identity to the amino acid sequence from amino acids 23 to 408 of SEQ ID NO. 2; or (iii) a fragment of (i) or (ii) which is capable of cleaving xylan.

2. A polypeptide according to claim 1 wherein the variant (ii) has at least 80% identity to the amino acid sequence from amino acids 23 to 408 of SEQ ID No. 2 and/or the fragment of (iii) is at least 150 amino acids in length.

3. A polypeptide according to claim 1 or 2 which cleaves (1—>4) linkages or adjacent xylopyranosyl units in §-D-xylan.

4. A polypeptide according to any preceding claim which has arabinoxylanase and xylosidase activity.

5. A polypeptide according to any preceding claim which is obtainable from (a) a fungus; or 2 (b) an organism of the genus Talaromyces, optionally of the species ~ Talaromyces emersonii.

6. A polynucleotide comprising: (a) the nucleic acid sequence of SEQ ID No. 1 or a sequence encoding a polypeptide according to any preceding claim; (b) a sequence which is complementary to, or which hybridises under stringent conditions to, a sequence as defined in (a); © a fragment of at least 100 nucleotides of a sequence in (a) or (b); (d) a sequence having at least 70% identity to a sequence as defined in (a), (b) or (c); or (e) a sequence that is degenerate as a result of the genetic code to any one of the sequences as defined in (a) to (d).

7. A sequence according to claim 6 wherein the fragment in (c) is at least 200 bases in length and/or the identity in (d) is at least 80%. :

8. A polynucleotide according to claim 7 which comprises: (a) a sequence that encodes a polypeptide having xylanase activity, which is: 55 Amended Sheet — 2004-05-25 y

(1) the coding sequence from nucleotides 69 to 1224 of SEQ ID No. 1; 2) a sequence which hybridises selectively to the complement of sequence defined in (1); or 3) a sequence that is degenerate as a result of the genetic code with respect to a sequence defined in (1) or (2); or (b) a sequence complementary to a polynucleotide defined in (a).

9. A polynucleotide according to any of claims 6 to 8 which is a DNA sequence.

10. A vector comprising a polynucleotide sequence according to any one of claims 6 to 9.

11. A vector according to claim 10 which is an expression vector.

12. A vector according to claim 11, wherein the expression vector comprises a DNA sequence according to claim 9 operably linked to a regulatory sequence.

13. A host cell which comprises, as a heterologous sequence, a polynucleotide according to any of claims 6 to 9.

14. A host cell which expresses, as a heterologous protein, a polypeptide according to any of claims 1 to 5.

15. A host cell transformed with the DNA sequence of claim 8 or a vector of claim 10.

16. A process of producing a polypeptide according to any of claims 1 to 5, the : process comprising culturing a host cell as defined in any of claims 13 to 15 under conditions that provide for expression of the polypeptide.

17. A composition comprising a polypeptide according to any one of claims 1 to 5.

18. A composition according to claim 17 which further comprises a polypeptide having cellulase, endo-arabinanase, thamnogalacturonase or polygalacturonase activity.

19. A method of treating a plant or xylan-containing material, the method comprising contacting the material with a protein according to any one of claims 1 to 5 or a composition according to claim 17 or claim 18.

20. A method according to claim 19 wherein the treatment comprises degrading, hydrolysing or modifying xylan in the material or degrading or modifying plant cell walls. 56 Amended Sheet — 2004-05-25

21. A method according to claim 19 or 20 wherein the treatment comprises cleaving of xylopyranosyl or B-D-xylan subunits and/or the material comprises a plant, plant pulp, plant extract or an edible foodstuff or ingredient therefor.

22. A processed material obtainable by contacting a plant or xylan-containing material with a polypeptide according to any one of claims 1 to 5 or a composition according to claim 17 or 18, or which results from a method according to any of claims 19 ~ to2l. :

23. A method according to any of claims 19 to 21 which reduces the viscosity of the material, degrades or hydrolyses xylan contained in the material or improves clarity or filterability of the material.

24. Use of a polypeptide according to any one of claims 1 to 5 or a composition according to claim 17 or claim 18 in a method of treating plant material, improving filterability and/or reducing viscosity of xylan-containing liquids, improving filterability or clarifying alcoholic liquids or fruit or vegetable juices, hydrolysing agricultural residues, in recycling materials in paper making for thickening foodstuffs and/or extracting desirable materials, processing plant pulp, juice or extract, improving loaf volume, bread quality or reducing the stickiness of dough.

25. Use according to claim 24, wherein the alcoholic liquids are beer or wine.

26. Use according to claim 24, wherein the recycling materials contain paper.

27. Use according to claim 24, wherein the desirable materials are coffee, plant oil or starch.

28. A feed, food or foodstuff comprising a polypeptide according to any one of claims 1 to 5.

29. A feed, food or foodstuff according to claim 28, which is an animal feed, food or foodstuff.

30. The use of a polypeptide according to any of claims 1 to 5 in brewing, beer or wine-making, distilling, recycling, biomethanation, dental hygiene, leather treatment, paper manufacture, fruit or vegetable juice or extract treatment, textile treatment or manufacture, baking or bread making, treating flower bulbs, preparation of food or foodstuffs or in an animal feed.

31. A food or foodstuff according to either of claims 28 or 29 which is an alcoholic beverage, bread, dough or tea. 57 Amended Sheet — 2004-05-25

32. A transgenic organism, comprising a cell according to any of claims 13 to

33. A transgenic organism according to claim 32, which is a plant or part thereof. 58 Amended Sheet — 2004-05-25