RU2291901C2 - Polypeptide and composition having xylanase activity, polypeptide application, polynucleotide encoding polypeptide, expression vector containing polynucleotide, method for production of polynucleotide, and method for treatment of plant or xylan-containing material - Google Patents

Abstract

FIELD: food processing industry, biotechnology.

SUBSTANCE: invention relates to polypeptide having xylanase activity which is capable to decompose of cellulose extracts and plant materials. In another embodiment disclosed is polynucleotide encoding said polypeptide, as well as composition containing polypeptide of xylanase activity. Abovementioned polypeptide is useful in treatment of plant or xylan-containing material for preparation of bread and animal feedstuff.

EFFECT: enhanced assortment of xylanases useful in industry.

23 cl, 10 tbl, 12 ex

Description

FIELD OF THE INVENTION

The present invention relates to new xylanases, such as xylanases from Talaromyces, and their use in the decomposition of xylan in cellulose. Xylanases are used in bread baking, animal feed (to improve feed processing) and in paper production.

BACKGROUND OF THE INVENTION

The composition of the cell walls of plants is complex and variable and contains several carbohydrate biopolymers. Polysaccharides are mainly in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicelluloses (including various β-xylan chains, such as xyloglucans), pectin and lignin. The most common 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 β-1,4-linked D-xylopyranosyl units optionally substituted with side chains such as arabinose and / or glucuronic acid residues. This structure represents

→ 4) -β-D-Xylp- (1 → 4) -β-D-Xylp (2 ← 1A) - (1 → 4) -β-D-Xylp- (1 → 4) -β-D-Xylp (3 ← 1B) - (1 →

(Xylp = xylopyranosyl link; A = α- (4-O) -methyl- (D-glucuronopyranosyl link), sometimes acetyl; and B = α- (L-arabinofuranosyl link), sometimes acetyl).

Xylans can make up more than 30% of the dry weight of land plants. Therefore, xylans are an important component of natural source materials that are used in industrial production, from baking, improving animal feed to making paper.

There is a major difference between monocotyledonous plants (for example, cereals and herbs) and dicotyledonous plants (for example, clover, rapeseed and soybean) and between the seed and vegetative parts of plants. Monocotyledonous plants are characterized by the presence of arabinoxylan complexes as the main framework of hemicellulose, and the xyloglucan complex is the main structure of hemicellulose in dicotyledonous plants. In dicotyledonous plants, higher concentrations of pectin were found than in monocotyledonous plants. Seeds usually have a high content of peptic substances, but a relatively low content of cellulose tissue.

Cellulose degrading enzymes are used to process plant material in food products, as well as when using fodder or as food or feed additives due to their ability to act on the main components of the cell wall.

Most cellulose degrading enzymes suitable for industrial use are xylanases with relatively low molecular weight and moderate stability at high temperatures. However, for certain applications, it is desirable to use xylanases with relatively high thermal stability. If xylanases are used as feed additives for animals, then high thermal stability is preferable, since high temperatures are used in the process of granulating animal feed.

SUMMARY OF THE INVENTION

The present invention relates to a novel xylanase having the ability to cleave β-D-xylan, which is present in plant material. This xylanase may also have the ability to hydrolyze arabinoxylan (or have arabinoxylanase activity) and aryl-β-D-xylopyranoside (or have xylosidase activity).

Accordingly, the present invention relates to a (isolated) β-xylanase polypeptide comprising:

(i) the amino acid sequence of SEQ ID No: 2; or

(ii) a variant of the sequence (i) having the ability to cleave β-D-xylan; or

(iii) a fragment of sequences (i) or (ii) having the ability to cleave β-D-xylan.

In accordance with another aspect, the invention relates to a polynucleotide containing:

(a) a nucleic acid sequence of SEQ ID No.1 or a sequence encoding a polypeptide of the invention;

(b) a sequence complementary or hybridizing to the sequence defined in (a);

(c) a fragment of the sequence (a) or (b);

(d) a sequence at least 60% identical to the sequence defined in (a), (b) or (c);

or

(e) a sequence that is genetically degenerate with respect to any of the sequences defined in (a) to (d).

The invention also relates to:

- a vector (eg, expressing) comprising a polynucleotide according to this invention and which may have the ability to express a polypeptide according to this invention;

- a cell line comprising the vector of the invention;

- a method for producing a polypeptide according to this invention, which includes maintaining the cell line according to this invention under conditions suitable for expression of the polypeptide, and, if necessary, isolating the polypeptide;

a method for decomposing β-D-xylan, the method comprising reacting a material containing β-D-xylan with a polypeptide of the invention; and

- a method for identifying a substance that modulates the activity of xylanase, which consists in the interaction of the polypeptide of this invention with the test substance in the presence of β-D-xylan and observing the activity or detecting any change in activity.

A brief description of the sequences

SEQ ID No.1 is a DNA sequence encoding a xylanase of the present invention from Talaromyces emersonii;

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

SEQ ID Nos. 3 and 4 synthetic PCR primers hybridizing with SEQ ID No.1.

DETAILED DESCRIPTION OF THE INVENTION

A. Polynucleotides

The present invention relates to a polynucleotide (e.g., isolated and / or purified) encoding a polypeptide of the invention. The present invention thus relates to a polynucleotide encoding xylanase, the amino acid sequence of which is shown in SEQ ID No.2 (such as, for example, a mature sequence of amino acids 23-408). Further, the present invention relates to a polynucleotide encoding a polypeptide having substantial homology of the amino acid sequence to the amino acid sequence shown in SEQ ID No.2. Also included is a polynucleotide selected from:

(a) a polynucleotide comprising a nucleotide sequence (eg, polynucleotides 69-1224) shown in SEQ ID No.1, or complementary to it;

(b) a polynucleotide comprising a nucleotide sequence having the ability (for example, selectively) to hybridize with the nucleotide sequence shown in SEQ ID No.1, or with a fragment thereof;

(c) a polynucleotide comprising a nucleotide sequence having the ability (for example, selectively) to hybridize with a nucleotide sequence that is complementary to that shown in SEQ ID No.1, or a fragment thereof; and / or

(d) a polynucleotide comprising a polynucleotide sequence that is genetically degenerate with respect to a polynucleotide as defined in (a), (b) or (c).

The polynucleotide of this invention also includes a polynucleotide that:

(a) encodes a polypeptide having xylanase activity, wherein the polynucleotide is:

(1) the coding sequence of SEQ ID No.1 (e.g., polynucleotides 69-1224);

(2) a sequence selectively hybridizing to a sequence that is complementary to that defined in (1); or

(3) a sequence that is genetically degenerate with respect to the sequence defined in (1) or (2); or

(b) is a sequence complementary to a polynucleotide as defined in (a).

References to SEQ ID No.1 may be replaced by a mature coding sequence (polynucleotides 69-1224), unless otherwise specified.

Hybridizing Sequences

The term “capable of hybridization” means that the target polynucleotide of this invention can hybridize with the nucleic acid used as a probe (for example, the nucleotide sequence shown in SEQ ID No.1, or a fragment or sequence complementary to it) at the level significantly higher than the background. The invention also includes nucleotide sequences encoding xylanase or its variants, as well as nucleotide sequences complementary to them. 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 more preferably a cDNA sequence. Typically, the polynucleotide of the present invention contains a continuous nucleotide sequence capable of hybridization under selective conditions with a coding sequence or a sequence complementary to (e.g., mature) the coding sequence of SEQ ID No.1. Such nucleotides can be synthesized in accordance with methods well known in the art. ¹ .

The polynucleotide of the present invention can hybridize to a coding sequence or to a sequence complementary to (e.g., mature) the coding sequence of SEQ ID No.1 at a level well above the background. Background hybridization may occur, for example, due to the presence of other cDNAs in the cDNA library. The signal level (for example, generated by the interaction between the polynucleotide of the present invention and the coding sequence or a sequence complementary to the coding sequence) is usually at least 10 times, preferably at least 100 times, higher than when interacting between other polynucleotides and (e.g., mature) coding sequence of SEQ ID No.1. The strength of the interaction can be measured, for example, using a radiolabeled probe, for example ³² P. Typically, selective hybridization can be performed under conditions of low stringency (0.3 M sodium chloride and 0.03 M sodium citrate at approximately 40 ° C), medium stiffness (e.g. 0.3 M sodium chloride and 0.03 M sodium citrate at about 50 ° C) or high stiffness (e.g. 0.3 M sodium chloride and 0.03 M sodium citrate at about 60 ° C). Hybridization can be performed under any suitable conditions known in this field ¹ , and as an instruction, low stiffness can be 2 × SSC at 55 ° C, average stiffness can be 0.5-1.0 × SSC at 60 ° C and high stiffness can be from 0.1 or 0.2 × SSC at 60 ° C or higher (for example, at 68 ° C), all at 0.5% SDS.

Modifications

Polynucleotides of the invention may be composed of DNA or RNA. They can be single or double stranded. They can also be polynucleotides that contain one or more synthetic or modified nucleotides. A number of different types of polynucleotide modifications are known in the art. These include methylphosphonate and phosphorothioate scaffolds and / or the addition of acridine or polylysine chains at the 3 ′ and / or 5 ′ ends of the molecule. For the purposes of the present invention, it should be understood that the polynucleotides described herein can be modified by any methods known in the art.

It should be understood that specialists in this field can, using conventional techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention, based on the frequency of use of the codons of any particular host organism, for example, in which the polypeptides of this invention are expressed.

The coding sequence (eg, mature) of SEQ ID No.1 may be modified by nucleotide substitutions, for example, from or to 1, 2 or 3 to 10, 25, 50, or 100 substitutions. Alternatively or additionally, the polynucleotide may be modified with one or more inserts, and / or deletions, and / or extension at one or both ends. The modified polynucleotide typically encodes a polypeptide having xylanase activity. Degenerate substitutions can be obtained and / or substitutions can be obtained, resulting in conservative amino acid substitutions in which a modified sequence is translated, for example, as discussed further below with respect to polypeptides.

Homologues

A nucleotide sequence capable of selective hybridization with (for example, a complementary sequence) the DNA coding sequence of SEQ ID No.1 (or nucleotides 69-1224) may have at least 70%, at least 75%, at least 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 can be in the region of at least 20, preferably at least 30 or 60, for example at least 100, at least 200, more preferably at least 300 adjacent nucleotides, or, optimally, along the entire length of SEQ ID No.1.

Any combination of the aforementioned degrees of homology and the minimum size can be used to determine the polynucleotides of the present invention, with more stringent combinations (i.e., with greater homology over a longer extent) are preferred. So, for example, one aspect of the present invention forms a polynucleotide representing at least 80% or 90% homology at 25, preferably 30 nucleotides, as well as a polynucleotide representing at least 90% homology at 40 nucleotides.

Homologues of a polynucleotide (or protein) sequence typically have at least 70% homology, preferably at least 80, 90%, 95%, 97% or 99% homology, for example in the region of at least 20, 25 , 30, 100 closer nucleotides (or amino acids). Homology can be calculated based on amino acid identity (sometimes referred to as “tight homology”).

For example, the UWGCG Package offers a BESTFIT program that can be used to calculate homology (for example, its default settings of ^{5 are used} ). The PILEUP and BLAST algorithms can be used to calculate homology or sequence alignment (such as determining equivalent or similar sequences, for example, with a default setting of ^6.7 ).

BLAST analysis software is available through the National Center for Biotechnology Information (http://www.ncbi.nhn.nih.gov/). This algorithm consists in the first determination of a pair of high-mark sequences (HSPs) by determining short words of length W in the desired sequence that either coincide or satisfy some positively estimated threshold mark T when aligned with a word of the same length in the database sequence. T is called the threshold mark of the neighboring word ^6.7 . These original matches of neighboring words act as a seed to initiate a search to find the HSPs containing them. Word matches are common in both directions along each sequence as much as the cumulative alignment mark can be increased. Elongations when words coincide in each direction stop when: the cumulative alignment mark falls out by the quantity X from its maximum value; the cumulative mark takes a value of zero or lower due to the accumulation of one or more residual alignments or upon reaching the end of any sequence. The BLAST W, T, and X algorithm parameters determine the sensitivity and speed of alignment. The BLAST program uses the default word length (W) 11, BLOSUM62 (B) 50 alignment matrix score ⁸ , expectation (E) 10, M = 5, N = 4, and a comparison of both circuits.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences ⁹ . One measure of similarity proposed by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence presumably similar to another sequence if the probability of the smallest sum in comparing the first sequence with 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 0.001.

Primers and probes

The polynucleotides of this invention contain and can be used as a primer, for example, PCR primer, an alternative amplification reaction primer, probe, or these polynucleotides can be cloned into vectors. Such primers, probes, and other fragments are at least or up to 20, 25, 30, or 40, for example, at least 25, 30, or 40 nucleotides in length. Usually they are up to 30, 40, 50, 60, 70, 100, 150, 200 or 300 nucleotides in length, or this number (even up to several nucleotides like 5 or 10 nucleotides) of a short (e.g. mature) coding sequence of SEQ ID No.1.

In general, primers will be prepared by synthetic methods, including the step-by-step preparation of a desired nucleotide sequence of one nucleotide per cycle. Techniques for doing this using automated technologies are readily available in the art. Examples of primers of this invention are given in SEQ ID Nos. 3 and 4.

Longer polynucleotides are usually obtained by recombinant methods, for example using PCR (polymerase chain reaction) cloning techniques. This includes obtaining a pair of primers (e.g., approximately 15-30 nucleotides) to the xylanase region to be cloned, contacting the primers with mRNA or cDNA derived from a target cell (e.g., yeast, bacterial, plant, prokaryotic or fungal ), preferably a Talaromyces strain, carrying out a polymerase chain reaction under conditions that amplify the desired region, isolate the amplified fragment (for example, by purifying the reaction mixture on an agarose gel) and restore amplified DNA. Primers can be designed to include recognition sites of the corresponding restriction enzyme, so that the amplified DNA can be cloned into the corresponding cloning vector.

Such techniques can be used to obtain the whole or part of the xylanase sequence described herein. Genomic clones corresponding to cDNA SEQ ID No. 1, or a xylanase gene containing, for example, intron and promoter regions are also included in this invention and can also be obtained in a similar way (for example, recombinant methods, PCR, cloning techniques), based on genomic DNA from cells of fungi, yeast, bacteria, plants or prokaryotes.

Polynucleotides or primers may contain a recognizable label, for example a radioactive or non-radioactive label. Suitable labels include radioisotopes, such as ³² P or ³⁵ S, enzyme labels, or other protein labels, such as biotin. Such labels can be added to the polynucleotides or primers of this invention and can be detected using known techniques.

Labeled or unlabeled polynucleotides can be used in nucleic acid-based studies to detect or sequence xylanase or its variants in a sample (e.g., fungi). Such detection studies mainly involve bringing a sample (e.g., fungal), (presumably) containing DNA, into contact with the probe or primer of this invention under hybridization conditions and detecting any duplexes formed between the probe and the nucleic acid in the sample . Such detection can be carried out using techniques such as PCR, or by immobilizing the probe on a solid support, removing a nucleic acid that does not hybridize with the probe in the sample, and then detecting a nucleic acid that hybridizes with the probe. Alternatively, the nucleic acid of the sample can be immobilized on a solid support, and the amount of probe bound can be determined.

The probes of the present invention can conveniently be packaged in the form of a kit for research in a suitable container. In these kits, probes can be bound to a solid support if the research format for which the kit is designed requires such binding. The kit may also contain reagents for processing the test samples, hybridization of the probe with the nucleic acid in the sample, control reagents, instructions and the like.

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

The polynucleotides of this invention also include sequence variants of SEQ ID No.1 that have xylanase activity. Variants may be formed by extensions, substitutions and / or deletions and may have the ability to cleave the β-D-xylan polymer.

Obtaining polynucleotides

Polynucleotides that do not have 100% identity with (for example, a mature coding sequence) SEQ ID No.1, but falling within the scope of the claims of the present invention, can be obtained in accordance with a number of methods. Thus, variants of the xylanase sequence described herein can be obtained, for example, by probing libraries of genomic DNA obtained from a number of organisms, for example, those indicated as sources of polypeptides of this invention. In addition, other fungal, plant, or prokaryotic xylanase homologs and such homologs, and fragments thereof, will generally be capable of hybridization with SEQ ID No.1. Such a sequence can be obtained by probing cDNA libraries or genomic DNA libraries from other species, and probing such libraries with probes containing the entire sequence or part of SEQ ID No.1 under medium or high stringency conditions (as described previously). Nucleic acid probes containing the whole sequence or part of SEQ ID No.1 can be used to probe cDNA libraries from other species, such as those described as sources for the polypeptides of this invention.

Interspecific homologues can also be obtained using degenerate PCR, which uses primers designed for target sequences within variants and homologues encoding conserved amino acid sequences. Primers may contain one or more degenerate positions and will be used under conditions of less stringency than that used to clone sequences with a single primer sequence against known sequences.

Alternatively, such polynucleotides can be obtained by site-directed mutagenesis of xylanase sequences or variants thereof. This may be applicable in cases where, for example, sequences require silent codon substitutions in sequences to optimize the predominance of codons for a particular host cell in which polynucleotide sequences are expressed. Other sequence changes may be required to introduce restriction enzyme recognition sites or to alter the properties or functions of polypeptides encoded by polynucleotides.

This invention relates to double-stranded polynucleotides, including the polynucleotide of this invention and complementary to it.

The present invention also relates to polynucleotides encoding the polypeptides of the present invention described below. Since such polynucleotides will be used as sequences for the recombinant production of the polypeptides of this invention, they do not need to be capable of hybridization with the sequence of SEQ ID No.1, although this is usually desirable. Otherwise, such polynucleotides may be labeled, used, and made as described above, if desired. DNA fragments can be prepared using PCR techniques with specific primers. ^33.34

B. Polypeptides

The present invention relates to (for example, substantially purified and / or isolated) xylanase and its variants. The polypeptides of this invention may essentially consist of the amino acid sequence of SEQ ID No.2, or part thereof (such as a mature sequence from positions 23 to 408), or a variant thereof. Polypeptides can also be encoded by the polynucleotides of this invention, as described above. References to SEQ ID No.2 can only be replaced by a mature sequence (residues Ala ²³ to Leu ⁴⁰⁸ ), unless the context requires otherwise.

The polypeptides of this invention can be active against both arabinoxylan and aryl-β-D-xylosides (such as those having arabinoxylanase and xylosidase activity).

The polypeptide of this invention may be in isolated or substantially purified form. It is understood that the polypeptide can be mixed with carriers or diluents that will not interfere with the intended goals and / or function of the polypeptide, and yet will be considered substantially isolated. Typically, a polypeptide is included in a preparation in which more than 20%, for example more than 30%, 40%, 50%, 80%, 90%, 95% or 99% by weight of the polypeptide in the preparation is the polypeptide of this invention. These drugs are relatively pure compounds: for some applications, the polypeptide can be up to 10%, 5%, 2%, 1%, or even not more than 0.5% of the composition. Conventional methods for purifying and / or synthesizing the proteins of this invention ¹ can be used. For some formulations (for example, for non-pharmaceutical use), the amount of polypeptide present may be small, for example from 0.01 to 10%, for example from 0.1 to 5%, or 2%, or even from 0.2 to 1%.

Preferably, the polypeptide of this invention can be obtained from a microorganism that has a gene encoding an enzyme with xylanase activity. A more preferred microorganism is a fungus and an optimally filamentous fungus. Preferred organisms are thus Talaromyces, such as Talaromyces emersonii (e.g., CBS 393.64 or 814.70).

Activity

The polypeptide of this invention may have one or more of the following features, namely;

(1) possesses β-D-xylanase activity;

(2) has an optimum pH in the range from 2 to 6, for example from 3 to 5, optimally from 3.5 to 5.0;

(3) has optimal activity at a temperature of from 50 to 95 ° C, for example from 70 to 90 ° C, optimally from 75 to 85 ° C;

(4) has a molecular weight (deglycosylated) from 30 to 50 kDa, preferably from 35 to 45 kDa, optimally from 40 to 44 kDa or (glycosylated) from 50 to 75 kDa, preferably from 55 to 70 kDa, optimally from 60 to 66 kDa; and / or

(5) has an isoelectric point from 3.0 to 3.6.

The polypeptide may have an EC.3.2.1.8 activity. Preferably a Family 10 polypeptide (formerly F type).

"Xylanase activity" is defined as the ability to cleave cellulose or a β-D-xylan polymer (for example, found in plants, such as oats or barley). Therefore, this activity allows the cleavage of β-D-xylan, such as between adjacent xylopyranosyl end or non-end units. Preferably, cleavage occurs at the [xylopyranosyl (1 → 4) xylopyranosyl] bond. The polypeptide may preferably cleave between two adjacent (e.g., unsubstituted) units. Therefore, it may have endoactivity (i.e., be endoxylanase). The substrate polymer may or may not be substituted. It may also have exoactivity (i.e., be exoxylanase), such as cleavage of terminal xylopyranosyl units. Preferably, the polypeptide does not exhibit glucanase activity.

The polypeptides of this invention can also be active (or exhibit activity) against arabinoxylan. Arabinoxylan is a subgroup of xylan with L-arabino-furanosyl side chains linked to C-2 or C-3, or both, of the xylose residues of the main chain. Arabinoxylan has a CAS Registration No. 98513-12-3. It may have the structure of (1 → 4) -β-D-xylan with 3-linked α-L-arabinose branches. This type of xylan is commonly found in oat husk xylan.

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

Arabinoxylan is found in oat husk, and in this detailed description, polypeptide activity (EXU, as well as PAHBAH activity) is determined from wheat flour arabinoxylan (with an arabinose: xylose ratio of 41:59). The study of arabinoxylan (as a substrate) is described further in the examples.

The polypeptides of this invention may also have xylosidase activity, for example, have the ability to hydrolyze substituted (e.g., aryl) -β-D-xylosides (also known as xylopyranosides). For example, they may be capable of hydrolysis of 4-methylumbelliferyl-β-D-xylopyranoside (CAS Registration No. 6734-33-4, obtained from Sigma Chemical Co). This activity is the ability to release a fluorescent label from a substrate. It can also hydrolyze (be active on) 5-bromo-4-chloro-3-indoxyl-β-D-xylopyranoside (CAS Registration No. 207606-55-1). This combination of activity against both arabinoxylan and aryl-β-D-xylanoside is unusual ^36.37 and is a new combination of activities for a polypeptide having xylanase activity.

Variants and homologues

The polypeptide of this invention may contain the amino acid sequence shown in SEQ ID No. 2, or a substantially homologous sequence, or a fragment of one of the two sequences and may have xylanase activity. In general, the naturally occurring amino acid sequence shown in SEQ ID No.2 is preferred.

In particular, the polypeptide of this invention may contain:

a. the (mature) polypeptide sequence of SEQ ID No.2 (residues 23 to 408) or the whole sequence of SEQ ID No.2;

b. natural variant or its interspecific homologue; or

c. protein with at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or with at least 99% sequence identity to (a) or (b).

The variant may be one found in nature, for example in fungal, bacterial, yeast, or plant cells, and which may function substantially similarly to the protein of SEQ ID No.2, for example, have xylanase activity. Similarly, the interspecific protein homologue will be an equivalent protein that is found in nature in a different form and that can function as xylanase. Variants include allelic variants either from the same strain as the polypeptide of the present invention, or from another strain, but of the same genus, or of the same species.

Variants and varieties of homologues can be obtained by the following methods described here to obtain the polypeptide SEQ ID No.2, the implementation of such methods on an appropriate source of cells, for example a bacterial, yeast, fungal or plant cell. It is also possible to use a probe as defined above to probe libraries composed of yeast, bacterial, fungal, or plant cells to produce clones that include variants or interspecies homologs. Clones can be influenced by conventional techniques to produce the polypeptide of the invention, which will then be prepared by recombinant or synthetic methods known per se.

The polypeptide of this 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 least 97% or at least 99% sequence identity, for example, over a region of at least 40, 60, 100, 150, 200, 300, or 400 amino acids or the entire length of SEQ ID No.2.

The sequence of the polypeptide SEQ ID No.2 and variants and interspecific homologs can therefore be modified to provide the polypeptides of this invention. Can be made, for example, from 1, 2 or 3, or up to 10, 20, 30, 50 or 100 amino acid substitutions. The same number of deletions or insertions can be made. These changes can be made outside the areas necessary for the functioning of the polypeptide, and can also still result in an active enzyme. The modified polypeptide mainly retains xylanase activity.

The polypeptides of this invention include fragments of the aforementioned full-sized polypeptides and their variants, including fragments of the sequence shown in SEQ ID No.2. Such fragments usually retain xylanase activity. Fragments may be at least 50, 60, 70, 80, 100, 150, 200, or 250 amino acids in length, or be less than the full length of the sequence of amino acids (shown in SEQ ID No.2). Fragments or variants include or represent a β-D-xylan binding region or a β-D-xylan cleavage region.

The polypeptides of this invention, if necessary, can be obtained by synthetic methods, although they are usually obtained recombinantly, as described below. They can be modified, for example, by adding histidine residues or a T7 tag in order to facilitate their identification or purification, or by adding a signal sequence that facilitates their secretion from the cell.

The term "variants" refers to polypeptides that may have the same basic property or basic biological function as xylanase, and include allelic variants. The main property of xylanase is that it is an enzyme that can cleave 1 → 4 bonds in β-D-xylan. A polypeptide having the same basic property as xylanase can be identified by studying cellulose degradation, as described below.

Variants of SEQ ID No.2 also include sequences that differ from SEQ ID No.2, but which do not necessarily come from natural xylanase. These options can be described as having% homology to SEQ ID No.2 or having a number of substitutions for this sequence. Alternatively, the variant may be encoded by a polynucleotide that hybridizes with SEQ ID No.1.

Variants may be similarly defined for variants of SEQ ID No.1. Therefore, the variants may contain a different sequence originating from other strains of Talaromyces. Other options can be established from other strains of Talaromyces by searching for xylanase activity and cloning and sequencing as before. Options may contain deletions, modifications or additions of single amino acids or groups of amino acids within the protein sequence, while the peptide retains the basic biological function of xylanase.

Conservative substitutions can be made, for example, according to the following table 10. The amino acids of the same block in the second column and preferably in the same line in the third column can be replaced by each other. Preferably, the substitutions do not affect the folding or activity of the polypeptide.

Table 10 ALIPHATIC Non-polar G a p I L V Polar uncharged C S T M N Q Polar charged D e K r AROMATIC H F W Y

Modifications

The polypeptides of this invention can be chemically modified, for example, modified post-translationally. For example, they can be glycosylated (one or more times, with the same or different sugars) or contain modified amino acid residues. They can also be modified by adding histidine residues (to facilitate their purification) or by adding a signal sequence (to facilitate incorporation into the cell membrane). The polypeptide may have one or more (N) amino or (C) carboxy-terminal extensions, such as amino-terminal methionine residues, a (small) linker peptide of up to about 20-25 residues, or a (small) purification extension, such as polyhistidine or a T7 label, an antigenic epitope, or (e.g., maltose) binding domain ¹⁴ (e.g., at the C-terminus). These extensions may or may not be attached via the linker.

The polypeptide of this invention may be labeled with a detectable label. A detectable label may be any suitable label that allows the detection of a polypeptide. Suitable labels include radioisotopes, for example ¹²⁵ I, ³⁵ S, enzymes, antibodies, polynucleotides and linkers, such as biotin.

Polypeptides can be modified to include non-natural amino acids or to increase the stability of the polypeptide. In the case when proteins or peptides are obtained by synthetic methods, such amino acids can be introduced during the synthesis. Proteins or peptides can also be modified by the following synthetic or recombinant treatment.

The polypeptides of this invention can also be obtained using or incorporating D-amino acids in them. In such cases, amino acid residues can be linked using the usual N-C sequence as described in this application.

A number of side chain modifications are known in the art and can be made with the side chains of the proteins or peptides of the present invention. Such modifications include, for example, amino acid modifications by reductive alkylation by reaction with an aldehyde followed by reduction of NaBH ₄ , amidination with methylacetimidate or acylation with acetic anhydride.

The sequences of the present invention can also be used as starting material for the construction of "second generation" enzymes. “Second generation” xylanases may be those modified by mutagenesis (eg, site-directed mutagenesis) that have properties different from those of wild-type xylanases or recombinant xylanases, for example, obtained in accordance with the present invention. For example, the temperature or optimum pH, specific activity, affinity for the substrate, or thermal stability can be changed to better suit the application in a particular process.

Amino acids necessary for the xylanase activity of this invention and therefore preferred substitutional objects can be established in accordance with techniques known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis ¹⁰ . In the latter technique, mutations are introduced for each residue in the molecule and the resulting mutant molecules are tested for biological activity (e.g., xylanase activity) to determine the amino acid residues that are important for the activity of the molecule. Plots of enzyme-substrate interaction can be established by analyzing the crystal structure, determined by such methods as nuclear magnetic resonance, crystallography or photoaffinity labeling ^{11, 12, 13} or molecular modeling.

The use of yeast or fungal host cells is expected to provide post-translational modifications (e.g., proteolytic processing, myristylation, glycosylation, truncation and phosphorylation of tyrosine, serine or threonine) as potentially necessary to impart optimal biological activity to the recombinant expression products of this invention.

The polypeptides of this invention can be obtained as they are outside their natural cellular environment. Thus, they can be substantially isolated or purified, as discussed above, or found in cells in which they are not found in nature, for example in cells of other species of fungi, animals, yeast or bacteria.

C. Recombinant aspects

The invention also relates to vectors containing a polynucleotide of the invention, including cloning and expression vectors, and methods for culturing, transforming or transfecting such vectors in an appropriate host cell, for example, under conditions under which the expression of a polypeptide of the invention occurs. Also provided are host cells comprising a polynucleotide or a vector of the invention, wherein the polynucleotide is heterologous to the genome of the host cell. The term “heterologous” usually in relation to a host cell means that this polynucleotide does not occur in the genome of the host cell in nature or that this polypeptide is not produced by this cell in nature. 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, the genus Aspergillus.

Polynucleotides of the invention may be included in a recombinant replicable vector, for example, a cloning or expression vector. This vector can be used to replicate a nucleic acid in a compatible host cell. Therefore, in a further embodiment, this invention relates to a method for producing polynucleotides of this invention by introducing a polynucleotide of this invention into a replicable vector, introducing this vector into a compatible host cell and growing the host cell under conditions that allow replication of this vector. This vector can be isolated from the host cell. Suitable host cells are described below in connection with expression vectors.

Vectors

The polynucleotide of the invention may be inserted into an expression cassette. The vector into which the expression cassette or polynucleotide of the invention is inserted may be any vector that is conveniently subjected to recombinant DNA techniques, and the choice of vector will often depend on the host cell into which it is inserted. So, a vector can be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal object whose replication is independent of chromosome replication, for example, a plasmid. Alternatively, the vector, after being introduced into the host cell, can be integrated into the genome of the host cell and replicated along with the chromosome (s) into which it has been integrated.

Preferably, the polynucleotide of the invention in a vector is operatively linked to a regulatory sequence that is capable of providing expression of the coding sequence by a host cell, i.e. the vector is an expression vector. The term "operatively linked" refers to a compound where the described components are in a relationship that allows them to function in their intended way. A regulatory sequence, such as a promoter, enhancer or expression control signal, “operably linked” to a coding sequence, is positioned so that expression of the coding sequence is achieved under conditions compatible with the control sequences, or the sequences are arranged so that they function in accordance with their by destination, for example, transcription begins with the promoter and continues through the DNA sequence encoding the polypeptide.

The vector may be a plasmid, cosmid, viral or phage vector, typically provided with a replication origin, optionally a promoter for expression of the polynucleotide, and optionally an enhancer and / or promoter regulator. A terminator sequence may be present; there may also be a polyadenyl sequence. A 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 can be used in vitro, for example, to obtain RNA, or used to transfect or transform a host cell. They may contain 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 operatively linked to expression signals that are capable of driving expression of the DNA sequence in the host cells. Transformation techniques that are well known to those skilled in the art ^3.4 are available for transforming a suitable host with an expression construct. The expression construct can be used to transform the host as part of a vector that carries a selectable marker, or the expression construct can be co-transformed as a separate molecule along with a vector that carries a selectable marker. A vector may contain one or more selectable marker genes.

Preferred breeding markers ^15,16 , without limitation, include markers that make up for the defect of the host cell or endow with resistance to the drug. These include, for example, universal marker genes that can be used to transform most filamentous fungi and yeast, such as acetamidase or cDNA genes (amdS, niaD, facA genes or cDNAs from A. nidulans, A.oryzae, or A. niger), or genes that provide antibiotic resistance, such as resistance to G418, hygromycin, bleomycin, kanamycin, phleomycin or benomyl (benA). Alternatively, specially selected markers can be used, such as auxotrophic markers, which require the appropriate mutant host strains: for example, URA3 (from S. cerevisiae or similar genes from other yeast), pyrG or pyrA (from A. nidulans or A. niger) , argB (from A.nidulans or A.niger) or trpC. In a preferred embodiment, the selectable marker is removed from the transformed host cell after administration of the expression construct so as to obtain transformed host cells capable of producing a polypeptide that is free of selectable marker genes ^21,22 .

Other markers include ATP synthetase, subunit 9 (oliC), orotidine-5'-phosphate decarboxylase (pvrA), the bacterial gene for resistance to G418 (this can also be used in yeast, but not in fungi), the gene for resistance to ampicillin (E .coli), the neomycin resistance gene (Bacillus) and the E. coli uidA gene encoding β-glucuronidase (GUS). The vectors can be used in vitro, for example, to obtain RNA, or used to transfect or transform a host cell.

For most filamentous fungi and yeast, the vector or expression construct is preferably integrated into the genome of the host cell to produce stable transformants. However, suitable episomal vectors are also available for some yeasts into which an expression construct for stable, highly productive expression can be included, examples thereof include vectors derived from 2μ pKD1 Saccharomyces and Kluyveromyces plasmids, respectively, or vectors containing the AMA sequence (e.g., AMA1 from Aspergillus ^3.20 ). In the case where expression constructs are integrated into the genome of the host cells, the constructs are either integrated at arbitrary loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably contain a highly productive gene. A highly productive gene is a gene whose mRNA can comprise at least 0.01% by weight of the total cellular mRNA, for example, under induced conditions, or alternatively, a gene whose gene product can be at least 0.2% of the mass of total cellular protein, or in the case of a secreted gene product, can be secreted at a concentration of at least 0.05 g / L. A number of examples of suitable highly productive genes are provided below.

A vector or expression construct for a given host cell may contain the following elements operably linked to each other in the following order from the 5 ′ end to the 3 ′ end relative to the coding chain of a sequence encoding a polypeptide of the first invention:

(1) a promoter sequence capable of controlling transcription of a DNA sequence encoding a polypeptide in a given host cell;

(2) optionally, a signal sequence capable of controlling the secretion of a polypeptide from a given host cell into the culture medium;

(3) a DNA sequence encoding a mature and preferably active form of a polypeptide;

and preferably also

(4) a transcription termination region (terminator) capable of interrupting transcription below a DNA sequence encoding a polypeptide.

Below the DNA sequence encoding the polypeptide, there may be a 3'-untranslated region containing one or more transcription termination sites (e.g., terminator). The origin of the terminator is less important. A terminator may, for example, be native to a DNA sequence encoding a polypeptide. However, a yeast terminator is preferably used in yeast host cells, and a filamentous fungus terminator is used in filamentous host cells. More preferably, the terminator is endogenous to the host cell (in which the DNA sequence encoding the polypeptide is expressed).

Enhanced expression of the polynucleotide encoding the polypeptide of this invention can also be accomplished by selecting heterologous regulatory regions, for example, a promoter, secretory leader and / or terminator regions, which can serve to enhance expression and, if necessary, secretion levels of the protein of interest from the expression host and / or to provide an inducible control of the expression of the polypeptide of this invention.

In addition to the promoter native to the gene encoding the polypeptide of this invention, other promoters can be used to control the expression of the polypeptide of this invention. The promoter can be selected for its effectiveness in controlling the expression of the polypeptide of this invention in the desired expression host.

Promoters / enhancers and other regulatory expression signals may be selected compatible with the host cell for which the expression vector has been created. For example, prokaryotic promoters can be used, in particular suitable for use in E. coli strains. When expression is performed in mammalian cells, mammalian promoters can be used. Tissue-specific promoters, for example, hepatocytic cell-specific promoters, can also be used. Viral promoters can also be used, for example, long terminal repeats of the Moloney mouse leukemia virus (MMLV LTR), Routh sarcoma virus (RSV) promoter LTR promoter, SV40 (e.g. large T antigen) promoter, IE human cytomegalovirus promoter (CMV), virus promoters herpes simplex or adenovirus promoters, HSV promoters, such as IE (HSV promoters), or HPV promoters, in particular the upper regulatory region of HPV (URR). Yeast promoters include S. cerevisiae GAL4 and ADH promoters, nmt 1 and adh S. pombe promoters. Mammalian promoters include a metallothionein promoter that can be induced in response to heavy metals such as cadmium and β-actin promoters. Tissue-specific promoters, in particular endothelial or neuronal cell-specific promoters (e.g., DDAHI and DDAHII promoters), are particularly preferred.

A number of promoters ^15.16 can be used that are capable of controlling transcription in the host cells of this invention. Preferably, the promoter sequence is derived from a highly productive gene, as previously determined. Examples of preferred highly productive genes from which it is preferable to obtain promoters and / or which are contained in preferred predetermined loci of targets for integrating expression constructs include, but are not limited to, genes encoding glycolytic enzymes, such as triose phosphate isomerases (TFIs), glyceraldehydrogen phosphate dehydrogenases (HAA). ), phosphoglycerate kinases (FHA), pyruvate kinases (PIK or PKI), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamylases, proteases, xyl nases, cellobiohydrolases, β-galactosidases, alcohol (methanol) oxidases, elongation factors and ribosomal proteins. Specific examples of corresponding highly productive genes include, for example, the LAC4 gene from Kluyveromyces sp., Methanol oxidase genes (AOX and MOX) from Hansenula and Pichia, respectively glucoamylase (glaA) genes from A-niger and A.awamori, A.oryzae TAKA- amylase gene, A-nidulans gpdA gene, and T.reesei cellobiohydrolase genes.

Examples of strong constitutive and / or inducible promoters that are preferred for use in fungal expression hosts ^15,16,35 are promoters derived from fungal genes for xylanase (xlnA), phytase, ATP synthetase, subunit 9 (oliC), triose phosphatisomerase (tpi), alcohol dehydrogenase (AdhA) α-amylase (amy) amyloglucosidase (AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd).

Examples of strong yeast promoters are promoters derived from genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase and triose phosphate isomerase.

Examples of strong bacterial promoters are α-amylase and Spo2 promoters, as well as promoters from extracellular protease genes.

The native promoter of the xylanase-encoding gene can be transferred by a promoter that is regulated differently than the native promoter.

Promoters suitable for plant cells include nopalin synthase (nos), octopin synthase (ocs), mannopin synthase (mas), the small ribulose subunit (rubisco ssu), histone, rice actin, phaseolin, cauliflower mosaic virus and CMS 35 (19 CMV) and circovirus promoters. All of these promoters are readily available in this area.

The vector may also include sequences flanking the polynucleotide inducing RNA formation, which include sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences or viral genomic sequences. This will also allow the polynucleotides of this invention to be inserted into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector containing an expression cassette that the viral sequences are flanked can be used to produce a viral vector suitable for delivering polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex vectors ^18.19 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. Retroviral vectors, for example, can be used to stably integrate a polynucleotide that induces the formation of antisense RNA in the genome of a host cell. Replication-defective adenoviral vectors, in contrast, remain episomal and, therefore, allow transient expression.

The vector may contain a polynucleotide according to this invention, oriented in the antisense direction to ensure the formation of antisense RNA. This can be used to reduce, if desired, expression levels of the polypeptide.

Host Cells and Expression

A further aspect of the present invention relates to a method for producing a polypeptide according to this invention, which comprises culturing a host cell (e.g., transformed or transfected with an expression vector as described above) under conditions that allow expression (by this vector) of a coding sequence that encodes a polypeptide; and, optionally, isolating an expressed polypeptide. Polynucleotides of the invention may be included in a recombinant replicable vector, for example, an expression vector. The vector can be used to replicate a nucleic acid in a compatible host cell. Thus, in a further embodiment, the invention relates to a method for producing 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 that facilitate replication of the vector. The vector may be isolated 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 Sf9 cells and fungal (e.g. filamentous) cells.

Preferably, the polypeptide is obtained as a secreted protein, in which case the DNA sequence encoding the mature form of the polypeptide in the expression construct is operably linked to the DNA sequence encoding the signal sequence. Preferably, the signal sequence is a native (homologous) DNA sequence encoding a polypeptide. Alternatively, the signal sequence is a foreign (heterologous) DNA sequence encoding a 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 signal sequences derived from yeast α-factor genes. Similarly, a suitable signal sequence for filamentous fungal host cells is, for example, a signal sequence derived from filamentous fungal amyloglucosidase (AG) gene, for example, A. niger glaA gene. It can be used in combination with an amyloglucosidase (also called (gluco) amylase) promoter, as well as in combination with other promoters. Hybrid signal sequences may also be used in the context of the present invention.

Preferred heterologous secretory leader sequences are those derived from the fungus amyloglucosidase (AG) gene (glaA, both 18 and 24 amino acid variants, e.g., Aspergillus), the α-factor gene (yeast, e.g., Saccharomyces and Kluyveromyces), or the α-amylase gene ( Bacillus).

The vectors can be transformed or transfected with a suitable host cell, as described above, to ensure expression of the polypeptide of this invention. This process may include culturing a host cell transformed with an expression vector as described above under conditions that allow the vector to express a coding sequence encoding a polypeptide.

A further aspect of the invention therefore provides host cells transformed or transfected, or containing a polynucleotide or vector of the invention. Preferably, the polynucleotide is introduced into the vector for replication and expression of the polynucleotide. These cells are selected compatible with the specified vector and can be, for example, prokaryotic (eg bacterial), cells of fungi, yeast or plants.

A heterologous host may also be selected in which the polypeptide of this invention is formed in a form substantially free of other cellulose-degrading enzymes. This can be achieved by selecting a host that usually does not form enzymes such as, for example, Kluyveromyces lactis.

This invention relates to methods for producing a polypeptide according to this invention by methods of expression of a recombinant DNA sequence encoding this polypeptide. To this end, the DNA sequence of this invention can be used to amplify genes and / or exchange expression signals, such as promoters, secretory signal sequences, to achieve economical production of the polypeptide in a suitable homologous or heterologous host cell. A homologous host cell is a host cell that is a cell of the same species or variant of the species from which the DNA sequence originates.

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

A host cell can overexpress a polypeptide, and techniques for overexpressing are well known ³ . Thus, the host may have two or more copies of the encoding polynucleotide (and the vector, therefore, may have two or more copies, respectively).

Bacteria from the genus Bacillus are very suitable as heterologous hosts because of their ability to secrete proteins into the culture medium. Other bacteria suitable as hosts are bacteria from the genera Streptomyces and Pseudomonas. A preferred yeast host cell for expressing a DNA sequence encoding a polypeptide is a cell of the genera Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia and Schizosaccharomyces. More preferably, the yeast host cell is selected from the group consisting of species of Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var. Lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica and Schizosaccharomyces pombe.

However, fungal (e.g. filamentous) host cells are more preferred. 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. A filamentous host cell is more preferably a cell of the genera Aspergillus oryzae, Aspergillus sojae, Aspergillus nidulans, or species from the Aspergillus niger ²³ 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 ficumarium, ficumarium, ficumarium, reicium, 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 ^24.25 and Trichoderma species; bacteria, such as Bacillus species ^26.27 , for example Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Pseudomonas species; and yeast, such as, for example, Kluyveromyces ²⁸ species, for example Kluyveromyces lactis ^29, and Saccharomyces species, for example Saccharomyces cerevisiae.

The host cells of the present invention include plant cells, and the present invention therefore relates to transgenic organisms, such as plants and parts thereof, which contain one or more cells of the present invention. These cells may heterologously express the polypeptide of the invention or may heterologously contain one or more polynucleotides of the invention. Transgenic (or genetically modified) plants can therefore have a sequence (for example, stably) embedded in the genome encoding one or more polypeptides of the invention. Transformation of plant cells can be performed using known techniques, for example, using the plasmid Ti or Ri from Agrobacterium tumefaciens. This plasmid (or vector) may therefore contain the sequences necessary for infection of plants, derivatives of Ti and / or Ri plasmids can be used.

Alternatively, targeted infection of parts of plants, such as, for example, a leaf, root, or stem, can be performed. With this technique, an infected plant can be damaged, for example, by cutting with a razor or by pricking a plant with a needle or rubbing it with an abrasive. Then damage is inoculated with Agrobacterium. A plant, or part of a plant, can be grown in a suitable culture medium and develop into an adult plant. The regeneration of transformed cells into genetically modified plants can also be carried out using known methods, for example, selection of transformed germs using antibiotics and subculturing of these germs in a medium containing appropriate nutrients, plant hormones, and the like ¹⁷ .

Host cell culture and recombinant production

This invention also relates to cells that have been modified to express xylanase or its variants. Such cells include transient or preferably stable cell lines of higher eukaryotic cells, such as mammalian or insect cells, lower eukaryotic cells, such as yeast and (for example, filamentous) fungi cells, or prokaryotic cells, such as bacterial cells.

For the proteins of the present invention, transient expression in a cell line or on a membrane is also possible, such as, for example, in a baculovirus expression system. Such systems that are adapted for the expression of proteins in accordance with this invention are also included in the scope of the present invention.

In accordance with the present invention, the preparation of the polypeptide of the present invention can be carried out by culturing microbiological expression hosts that have been transformed with one or more polynucleotides of the present invention in a conventional enzymatic nutrient medium.

Recombinant host cells in accordance with this invention can be cultured using techniques known in the art. For each combination of promoter and host cell, culture conditions are available that facilitate expression of the DNA sequence encoding the polypeptide. After reaching the desired cell density or titer of the polypeptide, the culture is stopped and the polypeptide is isolated using known techniques.

The enzyme medium may include a known culture medium containing a carbon source (e.g., glucose, maltose, mellas, etc.), a nitrogen source (e.g., ammonium sulfate, ammonium nitrate, ammonium chloride, etc.), an organic source nitrogen (e.g., yeast extract, malt extract, peptone, etc.); and sources of inorganic nutrients (e.g., phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, a stimulant may be included (e.g., cellulose, pectin, maltose, maltodextrin, or xylogalacturonan).

Selection of an appropriate medium may be based on the choice of an expression host and / or based on the regulatory requirements of the expression construct. Such media are known to those skilled in the art. The medium may, if required, contain additional components that are more favorable for expression hosts than for other potentially contaminating microorganisms.

Fermentation can be performed over a period of 0.5-30 days. This may be a batch, continuous or batch process, acceptable at a temperature in the range from 0 to 45 ° C and, for example, at a pH between 2 and 10. Preferred fermentation conditions are a temperature in the range from 20 to 37 ° C and / or a pH between 3 and 9. Appropriate conditions are usually selected based on the choice of the expression host and the expressed protein.

After fermentation, if necessary, cells can be removed from the fermentation broth by centrifugation or filtration. After stopping the fermentation or after removing the cells, the polypeptide of the invention can then be recovered and, if necessary, purified and isolated by conventional methods.

D. The use of xylanase and methods for treating plants or cellulose (eg, xylan) -containing materials.

The polypeptides of the present invention having xylanase activity can be used to process fungal or plant material, including plant mass or plant extracts. For example, they can be used to process grain, vegetables, fruits, or from extracts. Conveniently, the polypeptide of this invention is combined with suitable (solid or liquid) carriers or diluents, including buffers, to form a composition / enzyme preparation. This polypeptide may be attached to or bound to a carrier, for example, immobilized on a solid phase. Therefore, the present invention provides in a further aspect a composition comprising a polypeptide of the present invention. This may be a form suitable for packaging, transportation and / or storage, preferably in cases where xylanase activity is maintained. The compositions may be a paste, liquid, emulsion, powder, cereal, granule, ball, or other form.

In addition, the composition may contain additional ingredients, such as one or more enzymes, for example pectinases, including endoarabinases and ramnogalacturonase, cellulases, (other) xylanases, galacturonases, mannanases and / or xyloglucanases. The polypeptide is usually stably formulated in either liquid or dry form. Typically, the product is formulated as a composition, which optionally includes, for example, a stabilizing buffer and / or preservative. The compositions may also include other enzymes capable of degrading plant material or cellulose, for example, other cellulases, for example (β-D-) glucanases. For certain applications, immobilization of the enzyme on a solid matrix or incorporation on or within the particles of a solid carrier may be preferred. The composition may also include a number of other enzymes that decompose plant material, such as cellulases or other pectinases.

The polypeptides and compositions of this invention can therefore be used in methods of processing plant material to decompose or modify cellulosic components (eg, xylan) of cell walls of plant or fungal material. Thus, in a further aspect, the present invention relates to a method for the decomposition or modification of plant cells, this method includes the interaction of a plant or fungal cell with a polypeptide or composition of the present invention.

The present invention also relates to a method for processing plant material, which comprises reacting the plant material with a polypeptide or composition of the invention to decompose or modify cellulose in a (plant) material. Preferably, the plant material is a plant mass or a plant extract, such as, for example, juices.

In particular, decomposition preferably includes the cleavage of xylan subunits of the cellulosic component of plant cell walls. Preferably, the plant material is cereal, vegetables, fruits, or vegetable or fruit pulp or extract. The present invention further provides treated plant material obtained by reacting the plant material with a polypeptide or composition of the invention.

The present invention also relates to a method for reducing the viscosity of plant extracts, which comprises reacting the plant extract with the polypeptide or composition of the present invention in an amount effective to decompose the cellulose (or xylan) contained in the plant extract.

Plant or cellulosic materials include plant pulp, plant parts, and plant extracts. In the context of the present invention, an extract from plant material is any substance that can be obtained from plant material by extraction (mechanical and / or chemical), treatment, or other separating techniques. The extract may be juice, nectar, base or concentrates obtained from them. Plant material may include, or be obtained from vegetables, such as carrots, celery, onions, beans or legumes (soybeans, soybeans, peas) or fruits, such as pome fruits or seeds (apples, pears, quinces, etc. .), grapes, tomatoes, citrus fruits (oranges, lemons, limes, tangerines), melons, plums, cherries, black currants, red currants, raspberries, strawberries, cranberries, pineapples and other tropical fruits, trees and their parts (for example, pollen from conifers) or cereals (oats, barley, wheat, corn, rice). This material (hydrolyzed) can also be agricultural waste, such as sugar beet pulp, corn cobs, wheat straw, shells (peanuts), or recycled materials such as paper (waste paper).

The polypeptides of this invention, therefore, can be used to process plant material, including plant pulp or plant extracts. They can also be used to process liquid or solid feed or edible food ingredients, or can be used to extract coffee, vegetable oils, starch, or as a thickener in foods.

Typically, the polypeptides of this invention are used as a composition / enzyme preparation, as described above. Typically, the compositions will be added to the plant pulp obtained, for example, by machining, such as crushed or ground plant material. Incubation of the composition with the plant will typically be carried out over a period of from 10 minutes to 5 hours, such as, for example, from 30 minutes to 2 hours, preferably for about 1 hour. The processing temperature is preferably 10-55 ° C, for example from 15 to 25 ° C, optimally about 20 ° C, and 10-300 g, preferably 30-70 g, optimally about 50 g of enzyme per ton of material to be processed can be used. All used enzyme (s) or their compositions can be added sequentially or simultaneously with plant pulp. Depending on the composition of the enzyme preparation, the plant material may first be soaked (for example, for mashed potatoes) or liquefied. Using the polypeptides of the present invention, processing parameters such as extraction efficiency, viscosity of the extract and / or quality of the extract can be improved.

Alternatively or in addition to the foregoing, the polypeptide of the present invention may be added to untreated juice obtained from pressed or liquefied plant flesh. Processing of the crude juice will be performed similarly for the vegetable flesh in terms of dose, temperature and holding time. In addition, other enzymes may be included, such as those previously discussed. Typical incubation conditions are described in the previous section. The raw juice, incubated once with the polypeptides of this invention, is then centrifuged or (ultra) filtered to give the final product.

After treatment with the polypeptide of this invention, the (final) product can be heat treated, for example, at 100 ° C for a period of time from 1 minute to 1 hour under conditions for partial or complete inactivation of the polypeptide (s) of the present invention.

A composition comprising a polypeptide of the invention can also be used during the preparation of fruit or vegetable purees.

The polypeptide of this invention can also be used in brewing, wine making, distillation or baking. In addition, it can be used in the manufacture of alcoholic beverages such as wine and beer. For example, it can improve filterability or transparency (beer, wort or wine). Protein can contribute to the removal of dissolved organic substances from the broth or culture medium, for example, in cases where the waste from a distillation plant of organic origin is bioconverted into microbiological biomass. Xylanase can improve filterability and / or lower viscosity in glucose syrups, such as from cereals obtained by thinning (for example, α-amylase).

In bread baking, the polypeptide can improve the structure of the dough, change its stickiness or elasticity, improve the loaf volume and / or structure of the bread crumb, or impart better structural characteristics, such as brittleness, crumble, or quality of the bread crumb. The polypeptide can be added in amounts from 100 to 3000, for example, from 150 to 2000, optimally from 200 to 1600 EXU / kg of flour.

Polypeptides are used in various industries due to xylanase activity. These areas may include not only alcohol production, but also in biomethylation, bread making and baking, in oral hygiene (for example, compositions for teeth or oral cavity), in the processing or manufacture of leather, in the manufacture of paper, pharmaceuticals, in tea, in the manufacture or processing of fabrics, and in the processing of waste. One aspect of the present invention is therefore a food or food product containing a polypeptide, such as alcoholic beverages, bread, dough or tea. The polypeptide may be formulated in suitable compositions for any of these uses. The polypeptide may be present in an aqueous composition (e.g., hot water), preferably with one or more fungicides, for treating plant material (e.g., bulbs), especially for testing parasitic insects, ticks and nematodes. Since the polypeptides of this invention have the ability to decompose xylan, they can be added to food or food (for example, consumed by people). The invention also includes pharmaceutical and veterinary compositions that comprise a polypeptide of the invention and pharmaceutically or veterinarily acceptable carriers.

The polypeptides of this invention can also exhibit antifungal activity. They may have the ability to decompose the cell walls of fungi and, therefore, can be used to lysis the cell walls of fungi, to open cells. This can release intracellular proteins. Thus, these polypeptides can be used to prepare yeast and / or mushroom extracts.

E. Animal feed

This invention additionally relates to food or animal feed formulations or additives containing one or more polypeptides of the present invention. The polypeptide may be present in the feed at a concentration different from its natural concentration. Preferred amounts are 0.6-35, such as 1.5-15, preferably 3-15 mg per kg of feed. Suitably, the polypeptide of the invention is in the feed of from 1,000 to 50,000 EXU / kg of feed, for example from 2,500 to 25,000 EXU / kg, optimally from 5,000 to 20,000 EXU / kg.

This invention also relates to a method for producing a feed composition for animals, this method includes adding to one (or several) food substance (s) or ingredient (s) containing (x) xylan, a polypeptide according to this invention. These polypeptides can be added to the animal feed composition separately from the feed substances or ingredients, individually or in combination with other feed additives. This polypeptide may be an essential part of one or more feed substances or ingredients.

The polypeptides of this invention can also be added to animal feed rich in cellulose to improve the destruction of the cell wall of the plant, leading to improved assimilation of animal plant nutrients. The polypeptides of this invention may be added to the feed or silage if pre-soaking or liquid feeding is preferred. Advantageously, the polypeptides of this invention can continue to degrade cellulose in feed in vivo. Mushroom-based polypeptides of the present invention, in particular, generally have a lower optimum pH and have the ability to release important nutrients into an acidic environment such as in an animal’s stomach. The invention thus also contemplates feeds (for example, for animals) or food products containing one or more polypeptides of the invention.

The polypeptides of this invention can also be used in the manufacture of milk substitutes from soybeans. These milk substitutes can be consumed by both humans and animals. A typical problem in the production of these milk substitutes is the high viscosity of the soybean pulp, which results in the need for undesirable dilution of the pulp to a dry solids concentration of 10 to 15%. An enzyme preparation containing the polypeptide of the present invention can be added to or during the processing of the slurry, allowing treatment to be carried out at higher concentrations (usually from 40 to 50%) of the dry solid. These enzymes can be used in the preparation of tasty products, for example, from soybeans.

This composition may additionally contain (in particular, in the case of formulations for use in animal feed) one or more ionophores, oxidizing agents, surfactants, “rumen protected” amino acids, enzyme enhancers or enzymes that can be formed naturally in the gastrointestinal tract intestinal tract of fed animals.

When added to ruminants or monogastric animals (e.g. poultry or domestic pigs) (including silage), the feed may contain cereals such as barley, wheat, corn, rye or oats, or cereal by-products such as wheat bran or corn bran, or other plant materials such as soybeans and other legumes. This enzyme (s) can (gut) significantly improve the destruction of the cell walls of plants, which leads to better absorption of plant nutrients by animals. And, as a result, the growth rate and / or processing of feed can be improved. The polypeptides of this invention can be added to the feed (directly or as an additive or component) or can be added instead of the processed feed component (e.g. cellulose / xylan).

These proteins can reduce the viscosity of the feed (containing xylan): the proteins can continue the hydrolysis of xylan in vivo. The proteins of the present invention, in particular, are applicable to animal feed, as they remain active under conditions of high acidity, such as in the stomach of animals.

A particularly preferred method for (exogenously) adding a modified xylanase is to add a polypeptide of the invention as a transgenic plant material and / or (for example, transgenic) seeds. Therefore, the polypeptide is synthesized by heterologous gene expression, for example, a gene encoding the desired enzyme can be cloned into a plant expression vector under conditions suitable for plant expression signals, for example, a tissue-specific promoter, such as a specific seed promoter. An expression vector containing a gene encoding a polypeptide can subsequently be transformed into plant cells, and transformed cells can be selected for regeneration into whole plants. The transgenic plants thus obtained can be grown and harvested, and plant parts containing a heterologous (given plant) polypeptide can be included in one of the compositions, either as such or after further processing. Conventional methods are known for (heterologous) expression of enzymes in (transgenic) plants, including methods for seed-specific expression of enzymes ³⁰ . This heterologous polypeptide may be contained in the seed of a transgenic plant, or it may be contained in other parts of the plant, such as roots, stems, leaves, wood, flowers, bark and / or fruits. The plant may be monocotyledonous or dicotyledonous. Suitable plants include cereals such as oats, barley, wheat, corn, and rice. Preferably, the polynucleotide of the invention is stably incorporated into the plant genome.

Adding a polypeptide in the form of transgenic plant material, for example, in transgenic seeds, may require processing the plant material to make the enzyme available, or at least improve its availability. Such processing techniques may include a variety of mechanical (e.g., grinding and / or grinding) techniques or thermomechanical treatments, such as extrusion or expansion.

The present invention, therefore, also relates to a method of promoting growth and / or processing of food in monogastric or non-ruminant animals, this method includes feeding the animals with the polypeptide of this invention. Suitable animals include farm animals, monogastric and / or non-ruminant animals such as pigs (or piglets), poultry (such as chickens, turkeys), calves, or aquatic (e.g., marine) animals (e.g., fish).

Studying cellulose degrading enzymes

Also within the scope of the present invention is the use of the polypeptides of the invention for screening methods for determining substances that can act as antagonists that can modulate xylanase. In general terms, such screening methods may include reacting a polypeptide of the invention with a test substance and then measuring the activity or incubating the polypeptide of the invention with a test substance and then detecting any change in xylanase activity. The active substances that bind to the polypeptide of the present invention can also be determined by binding studies.

An activity modulator can be established by reacting cells expressing a polypeptide of the invention with a test substance and observing an effect mediated by the polypeptides. Cells expressing the polypeptide may be in vitro, and preferably the study is carried out in vitro using cells expressing the recombinant polypeptide.

The studies and substrates described here have established and confirmed xylanase activity. These studies can be used to identify other enzymes that decompose cellulose, for example, with xylanase activity. The substrate that can be used for this study may contain xylan.

Another aspect of the present invention relates to a study to determine or detect a polypeptide that has the ability to degrade cellulose. The activity may be xylanase, or may be pectinlyase, polygalacturonase, esterase, cellulase, xyloglucanase, galactonase, arabinanase or rhamnogalacturonase. This study may include:

(a) providing, as a substrate for the candidate substance (usually a polypeptide), a suitable substrate (as described); and

(b) the interaction of the substrate with the candidate substance and detecting whether any xylanase activity products are formed.

The above studies can be used to determine the modulators of xylanase activity. Such substances can reduce the softening of fruits, which can give a better taste and improve the color of fruits and can also extend the shelf life and / or transport. Thus, these studies can be used to identify inhibitors of the polypeptides of this invention, which may be able to inhibit the softening of fruits.

Preferred features and characteristics of one aspect of the present invention are applicability to other aspects of mutatis mutandis.

The invention will now be described with reference to the following examples, which are intended to illustrate, but not to limit.

EXAMPLES

General techniques

Standard molecular cloning techniques, such as DNA isolation, gel electrophoresis, enzymatic restriction modifications of nucleic acids, Southern analyzes, transformation of E. coli, colony lifts and filter hybridization, etc., were performed using standard methods ^1,2 . Synthetic oligodeoxynucleotides were obtained from ISOGEN Bioscience (Maarssen, Netherlands). DNA sequence analyzes were performed on an Applied Biosystems 373A DNA sequencer according to the instructions.

DNA labeling and hybridization was performed according to ECL ^™ direct labeling of nucleic acid and detection systems (Amersham LIFE SCIENCE, Little Chalfont, England) or in accordance with standard radioactive labeling methods ¹ .

EXAMPLE 1: isolation of RNA from T. emersonii and cDNA synthesis

T.emersonii strain CBS 393.64 was fermented under xylan-inducing conditions. At certain points in time, mycelium and culture supernatant were collected by filtration using Miracloth filtration wrap. The mycelium was washed well with demineralized water and squeezed into paper towels to remove excess water. The mycelium of selected times (based on measurements of cellulase in culture supernatants) was immediately frozen in liquid nitrogen and ground into a fine powder using a mortar and pestle. The resulting powder was transferred into a sterile 50 ml tube and weighed: for every 1-1.2 g of mycelium, 10 ml of TRIzol reagent (Gibco / BRL) was added (maximum 25 ml per tube). The mycelium powder was immediately dissolved by vigorous stirring (vortex, 1 min), followed by 5-min incubation at room temperature with periodic stirring. 0.2 (original TRIzol) of chloroform volume was added (therefore, 2 ml for every 10 ml of the originally used TRIzol), mixed by vigorous rotation and left at room temperature for 10 minutes. Subsequently, the mixture was centrifuged at 4 ° C, 6000 g for 30 minutes. The upper aqueous phase was transferred to a clean tube and all RNA was precipitated by adding 0.5 (starting TRIzol) volume of isopropyl alcohol (therefore, 5 ml of isopropyl alcohol for every 10 ml of TRIzol originally used). After 10 minutes of precipitation at room temperature, RNA was recovered by centrifugation for 30 minutes at 6000 g. After removal of the supernatant, the RNA pellet was washed with one volume of 70% ethanol. After ethanol was removed, the RNA pellet was air dried. The dried RNA pellet was dissolved in 3 ml of GTS buffer (100 mM Tris-Cl, pH 7.5, 4M guanidine thiocyanate, 0.5% sodium lauryl sarcosinate). 10 μl of RNA solution was used to determine the quality and concentration of nucleic acids.

Northern analysis ^{3 was} performed and the isolated RNA was further purified. ^1.3 A modified protocol (using pressureless flow instead of centrifugation) of the PHARMACIA purification kit (Cat no. 27-9258-02) was used to isolate mRNA. ³ For the synthesis of cDNA, STRATAGENE cDNA Synthesis KIT was used in accordance with the manufacturer's instructions, with the exception of a number of optimality criteria for using pGBFIN vectors with significant changes, as described previously. ³

The amount of synthesized cDNA was evaluated by TCA precipitation and subsequently analyzed by alkaline agarose gel electrophoresis. ³

EXAMPLE 2: Obtaining cDNA libraries from T. emersonii mRNA

The cDNA pool obtained in Example 1 was blunted, ligated with adapters, and digested with restriction enzyme. ³

Cloning of cDNA into the pGBFIN-11 ³ expression vector requires the presence of an EcoRI site at the 5 ′ and XhoI site at the 3 ′ end of the cDNA. Therefore, the primer first chain oligonucleotides used and the adapter sequences (Pharmacia) were selected according to the preconditions set for the expression vector.

The resulting cDNAs were separated by size fractionation through a SEPHAROSE CL-2B matrix, based on size, the individual pools obtained were analyzed by non-denaturing gel electrophoresis. ³ Two pools of cDNA obtained by clipping at 0.5 TPN and 1.0 kb respectively, were selected to construct a cDNA library in pGBFIN-11. For pGBFIN-11, a pool of two-chain split (EcoRI-XhoI) pGBFIN-11 vector was prepared (background ligation <1%). Selected cDNA pools were ligated into the pGBFIN-11 vector and bacterial cells were transformed into E. coli XL10-Gold to generate two primer cDNA libraries. The transformation frequencies of the two pools were> 1.0 × 10 ⁶ .

Clones were randomly selected from the fraction of both E. coli cDNA libraries and plasmid DNA was isolated. Analysis of this plasmid DNA showed that both cDNA libraries had a percentage insert between 90 and 95%.

In addition, colony lifts were carried out from the generated filters library fraction, subsequently hybridized with the T.emersonii gpdA gene encoding the glyceraldehyde-3-phosphate dehydrogenase gene. Next, plasmid DNA was isolated and, by restriction analysis, it was shown that all plasmids contained single inserts in the correct orientation. Sequencing of the 5 'ends of cDNA within T.emersonii gpdA showed that the plasmid content was> 85% of the total length.

EXAMPLE 3: Transformation of an Expression Library in A.niger

DNA was isolated from the E. coli cDNA library as previously described. Total plasmid DNA was digested for 4 hours at 37 ° C with NotI to remove plasmid sequences derived from E. coli. After purification, DNA was dissolved in sterile demineralized water.

³ multiple transformations of A.niger DS2978 were performed using from 1.5 × 10 ⁷ to 3.0 × 10 ⁷ protoplasts and 10 μg of plasmid DNA per transformation. Transformants were selected by the presence of an amdS selection marker by growing on acetamide as the sole nitrogen source. Since both the amdS selectable marker and the cDNA expression cassette are present on the integrating fragment, growth on acetamide is an indication of the presence of the cDNA expression cassette.

After approximately 7-10 days of incubation at 30 ° C, 10,000 transformants were purified: Aspergillus niger transformants were transferred automatically (Flexys ^™ colony picker automater) from the transformation plates to 96-well MTP Master Plates (MPs) containing 150 μl per well solidified selective medium (SM) (per 1000 ml: 0.52 g KCl, 1.52 g K ₂ HPO ₄ , 0.52 g MgSO ₄ , 20 g glucose, 1 g acetamide, 0.1 M MES buffer, 15 g agar, 1 ml of a trace element solution (containing per liter: 2.2 g of ZnSO ₄ / 7H ₂ O, 1.1 g of H ₃ BO ₃ , 0.5 g of FeSO ₄ / 7H ₂ O, 0.17 g of CoCl ₂ / 6H ₂ O , 0.16g CuSO ₄ / 5H ₂ O, 0.15g NaMoO ₄ / 2H ₂ O, 5,0 g EDTA, pH 6,5) pH 5,5. Transformants growing and SM for 5 days at 34 ° C. And thus the generated group MPs was used to inoculate MTPs for cultivation and subsequent identification of enzyme and duplicate plates (BPs) cDNA library stored at -80 ° C.

EXAMPLE 4: Analysis of the expression library T.emersonii

5-day-old sprouts of MPs were used as a replicated control and replicas were placed on plates with fresh selective medium containing 0.075% AZCL-xylan (containing per liter: 0.52 g KCl, 1.52 g K ₂ HPO ₄ , 0.52 g MgSO ₄ , 20 g glucose, 1 g acetamide, 0.1 M MES buffer, 15 g agar, 1 ml trace element solution (per liter: 2.2 g ZnSO ₄ / 7H ₂ O, 1.1 g H ₃ BO ₃ 0.5 g FeSO ₄ / 7H ₂ O, 0.17 g CoCl ₂ / 6H ₂ O, 0.16 g CuSO ₄ / 5H ₂ O, 0.15 g NaMoO ₄ / 2H ₂ O, 5.0 g EDTA , pH 6.5) pH 5.5, 0.75 g of AZCL-Xylan (Megazyme, Australia).

Once inoculated plates were incubated at 34 ° C for 48 hours and then for 6 hours at 65 ° C. Cups were viewed before and after high temperature incubation. Positive colonies exhibiting xylanase activity showed a blurred blue halo.

After the first selection, positive xylanase clones were seeded onto fresh SM medium and grown for 5 days at 34 ° C. The control plates thus obtained were then replicated on selective medium and on a medium containing 0.075% (w / v) AZCL-xylan (Megazyme). AZCL xylan plates were treated as previously described.

SM dishes were incubated for 48 hours at 34 ° C and then top-filled with oat husk xylan (5 g agarose, 0.5 g oat husk xylan (Sigma ref: X0627)) prepared in 1 liter of 50 mM phosphate buffer (pH 7). Upon hardening of the top layer of agar, the plates were placed for 4 hours at 65 ° C. To visualize the activity, the plates were stained with a solution of Congo red (10 g Congo red per 1 liter of phosphate buffer pH 7.0) for 15 minutes. The staining solution was discarded and the plates were washed with 1 M NaCl. This washing step was repeated twice. Positive colonies showed the formation of a pale halo of enlightenment against a background of (Congo) red. As a result, 9 xylanase positive clones were detected.

Xylanase-producing Aspergillus transformants, as defined in the xylanase plate assay, were grown in a fermentation shaker ³ . Samples of the medium were obtained after 5 days of fermentation and analyzed for xylanase activity as follows.

The supernatant (previously diluted if necessary) is diluted 5 times with 0.25 M sodium acetate buffer, pH 4.5. 20 μl of the diluted supernatant was transferred to microtiter plates and 50 μl of substrate (4% (w / v) Remazol Brilliant Blue RBB-Xylan (dissolved at 70 ° C. in demineralized water) was added and thoroughly mixed by pipetting up and down.The reaction mixture was incubated for 30 minutes at room temperature. The reaction was stopped by adding 200 μl of 96% ethanol and incubated for 10 minutes at room temperature. After completion of the reaction, the microtiter plates were centrifuged for 10 minutes at 2500 rpm in a Beckman GPK centrifuge at room temperature. 100 μl of the supernatant was transferred to a new microtiter plate and the absorbance of blue was measured spectrophotometrically at 620 nm in Anthosreader (Proton and Wilton). The specific activity was calculated by a calibration curve using a xylanase standard dissolved in 0.25 M acetate pH 4.5 buffer .

EXAMPLE 5: Genetic analysis of positive transformants

The positive (re-confirmed) transformants defined in Example 4 were grown in liquid medium, the mycelium was collected and the total (chromosome) DNA was isolated using the Puregene Isolation System (Biozym B.V.) to isolate DNA from filamentous fungi. DNA isolation and purification was carried out in accordance with the supplier's protocol, but slightly modified: stages 3 and 4 of protein precipitation were repeated.

Chromosomal DNA was used as a control in PCR using primers 12207 (SEQ ID No.4) and 11937 (SEQ ID No.3) for amplification of the insert (s) located in the expression cassette integrated into the chromosomal DNA.

PCR was directed to transformants in accordance with an adapted version of the known protocol ⁴ except that the resulting mycelium was subsequently treated with Glucanex ^™ (Novo Nordisk) at a concentration of 5 mg / ml instead of NOVOzyme.

PCRs contained eLONGase ^™ B buffer (Life Technologies, Breda, The Netherlands), dNTPs (200 μM each), 1 μl of eLONGase ^TM Enzyme Mix, 1-5 μl of control, and 10-30 pmol of each oligo, in a final volume of 50 μl. Optimal amounts of oligo were determined experimentally for each group obtained. On average, 10 to 30 pmol was used. The reactions were carried out under the following cycle conditions: 1 × (2 min) 94 ° C, 35 × (1 min 94 ° C, 1 min 55 ° C, 6 min 72 ° C), 1 × (7 min 72 ° C). Samples were placed on agarose gels for analysis of PCR products.

The PCR product thus obtained was subcloned into an E. coli pcr2.1 cloning vector (Invitrogen according to the supplier's instructions), plasmid pGBXEA-1. The E. coli strain that received the plasmid pGBXEA-1 was deposited with the Centraal Bureau voor Schimmelcultures, Baarn, the Netherlands, under accession number CBS 102183.

The subcloned PCR product was sequenced. The resulting nucleotide sequence of the coding region is depicted in SEQ ID No.1 and the deduced amino acid sequence of the protein in SEQ ID No.2. This protein was called XEA.

EXAMPLE 6: Characterization of xylanase Talaroymyces emersonii

The definition of endoxylanase unit (EXU) for this description.

A xylanase activity unit (EXU) is defined as the amount of an enzyme (endoI endo-1,4-β-xylanase from Asp.niger ³¹ ) that releases 4.53 μmol of recovered sugars (changed as xylose equivalents) per minute under study conditions. Research conditions include:

5 mg / ml arabinoxylan from wheat flour (Megazyme, Australia, 2/11 Ponderosa Parade, Warriewood NSW 2101) in 100 mM citrate buffer (pH 3.5), temperature 40 ° C, with a reaction time of 60 minutes. Reactions were stopped by the addition of 1 M NaOH. The determination was carried out colorimetrically at 420 nm after incubation of the samples with Fe-III-hexacyanide for 15 minutes in boiling water. Hexacyanoferrate reagent was prepared by dissolving 1.17 g of KFe (CN) and 19.5 g of anhydrous sodium carbonate in 1 liter of water.

Viscometric examination

In addition to the above absolute determination of xylanase activity, appropriate methods can be used that are used to lower the viscosity of wheat arabinoxylan solution (Megazyme, Australia, 2/11 Ponderosa Parade, Warriewood NSW 2101) by adding a certain amount of enzyme. Wheat arabinoxylan was dissolved in 0.425 M citrate buffer (pH 3.5) to a concentration of 8.3 mg / ml. The substrate was incubated at 55 ° C for 10 minutes. Then a small amount of the enzyme was added (in the range of 0.01-0.05 units / ml) and the reaction was carried out. After 60 minutes of reaction time, the viscosity of the sample was determined compared to the corresponding sample, which was incubated with the Aspergillus niger ³¹ endoxylanase standard of known ECE activity. The absolute activity in ECE for the standard was determined by the method of reducing sugars using Fe-III-hexacyanide, as described above. Viscosity was determined manually using a Haake viscous drop apparatus.

Analysis of the activity of reducing sugars

Enzymatic activity according to XPU determination was measured by determining reducing sugars using 4-hydroxybenzoic acid hydrazide (PAHBAH). One XPU activity is defined as the amount of enzyme required to release one μmol of reducing sugars generated per minute from wheat arabinoxylan at pH 5.0 and 60 ° C for 15 minutes using the D (+) xylose calibration curve. This modified known study ^{32 is} carried out with PAHBAH reagent as follows: 0.05 M sodium tricitrate, 0.1 M Na ₂ SO ₃ , 0.02 M CaCl ₂ , 0.5 M NaOH and 0.1 M p-hydroxybenzoic acid hydrazide (PAHBAH). The final pH was 12. A reagent containing PAHBAH in an alkaline solution was stored at room temperature, used for one day. The absorption was measured at 420 nm. The control was prepared by adding 100 μl of 0.1 M sodium acetate buffer instead of the enzyme solution. Xylanase activity was investigated by mixing 100 μl of a (diluted) enzyme solution with 400 μl of 0.35% wheat arabinoxylan (Megazyme) in 0.1 M acetate-sodium buffer (pH 5.0). Eppendorf tubes with substrate were preincubated for 5 minutes at 60 ° C. The reaction is started by adding an enzyme solution. After 15 minutes, the addition of 1.0 ml of PAHBAH reagent completes the reaction. Eppendorf tubes were heated for 5 minutes at 100 ° C and then cooled on ice. Samples were centrifuged at the appropriate speed to precipitate any solid materials, for example 1 minute at full speed in a Beckman Microfuge E. Absorption was measured at 420 nm. The control was prepared by adding 100 μl instead of the enzyme solution. The measurement interval is 0.01-0.1 XPU / ml.

Xylanase Purification

10.45 g of ammonium sulfate was added to 43 ml of cell-free broth and delivered to an Ethyl Sepharose ^TM column using a Äkta explorer 100 (Pharmacia Biotech) Column: 15 ETH Source (code 17-0146-01, Pharmacia Biotech) (D = 1 6 cm, l = 4.8 cm, V = 9.6 ml). The column was equilibrated with 100 mM sodium acetate and 40% saturated ammonium sulfate pH 5.0. Elution was carried out by a linear gradient, which was reduced to 100 mM sodium acetate (pH 5.0) in 20 column volumes. Fraction Size: 5 ml. Flow rate: 10 ml / min. Observed wavelengths: 280, 254, 214 nm. Fractions were checked for xylanase and analyzed by HPLC - by molecular size (SEC).

The purest xylanase fractions were added to the Sephacryl S200 column according to the following conditions. Equipment Äkta explorer 100 (Pharmacia Biotech). Column: HiPrep 16/60 Sephacryl S200 HR (Pharmacia Biotech). Balancing and elution was performed with 100 mM sodium acetate (pH 5.0). Flow rate: 1 ml / min. The fraction size is 4 ml. Observed wavelengths: 280, 254, 214 nm. The purity of the eluent fractions was analyzed by HPLC-SEC, SDS-PAGE and simple PAGE.

Protein concentration

Protein concentration was determined by measuring OD280. The (mature) xylanase from T. emersonii contains 11 Trp residues (positions 72, 108, 115, 121, 148, 300, 302, 308, 322, 377 and 385) and 23 Tyr residues (positions 36, 51, 109, 141, 146, 161, 167, 169, 174, 192, 193, 196, 200, 218, 281, 306, 316, 326, 332, 348, 395, 403 and 404). The calculated molar extinction coefficient was 89530 M ^-1 · cm ^-1 . The molecular weight was 41.637 g / mol. OD280 for 1 mg / ml XEA was 2.15.

The specific activity of xylanase from T. emersonii (XEA)

Specific activity was determined using the PAHBAH method of reducing sugars at 40 ° C and 60 ° C. The specific xylanase activity for XEA was 150 XPU and 500 XPU at the indicated two temperatures, respectively (table 11). Protein concentration was determined by analysis of OD 280.

Table 11 Temperature (° C) Specific Activity (XPU / mg) 40 150 60 500

N-terminal sequence

Found that the N-terminal amino acid sequence of purified mature XEA is:

Ala-Gly-Leu-Asn-Thr-Ala (in the mature sequence, the first Ala residue is Ala ²³ in SEQ ID No.2).

Isoelectric point (IET)

Equipment: Phast system (Pharmacia Biotech), IEF 3-9 Phastgels (Pharmacia Biotech). Gels were passed and stained (Coomassie) in accordance with standard Phast system protocols provided by manufacturers. The IET determined by isoelectric focusing using PhastGel IEF3-9 was approximately 3.3.

Molecular mass

SDS-PAGE electrophoresis was performed using the Phast system (Pharmacia Biotech), Phastgels (Pharmacia Biotech), SDS buffer strips / native buffer strips (Pharmacia Biotech).

Sample processing: one volume of buffer (500 mM Tris-HCl pH 6.8, 10% SDS, 0.1% bromophenol blue) was mixed with 4 volumes of the sample and boiled for 3 minutes. The gels were passed and stained (Coomassie or silver) in accordance with standard Phast system methods. The molecular weight based on SDS-PAGE using molecular weight markers (Pharmacia) was approximately 63 kD. The molecular weight calculated on the basis of the amino acid composition was 41,637 Daltons.

In addition, molecular weight was determined by permeation gel chromatography using molecular weight standards for gel filtration (BIORAD, cat. No.151-1901). The molecular weight determined by high-performance SEC was 42 kD. (HP-SEC was performed using a TSK G3000SW (cat. No 05103, Toso Haas) column. Samples were eluted in 0.1 M sodium phosphate (pH 7) at 1 ml / min at room temperature. Determination was performed at 280 nm). The high molecular weight, as observed by SDS-PAGE, seems overpriced. A likely cause of this is glycosylation of xylanase.

Deglycosylation

5 μl of purified enzyme (ca. 5 mg / ml) was mixed with 20 μl of 0.5% SDS and 25 μl of 1% mercaptoethanol. The mixture was boiled for 4 minutes. After cooling, 20 μl of N-glycosidase F (500 U / ml) and 20 μl of 3% Triton X-100 in 1 M phosphate-sodium buffer (pH 7.0) were added. It was then incubated overnight at 37 ° C and deglycosylation was analyzed by SDS-PAGE. The amino acid sequence offers 2 glycosylation sites (Asn ⁵⁶ and Asn ¹²³ , see SEQ ID No.2).

SDS-PAGE shows that xylanase-treated N-glycosidase F moves further and the molecular weight is lower than that of untreated or pre-treated (boiled with SDS and β-mercaptoethanol) xylanase. Thus, the unexpectedly high molecular weight observed with SDS-PAGE is possibly due to glycosylation.

pH and temperature profile

Cell-free broth was analyzed for xylanase activity at various pH and temperatures. Xylanase activity was analyzed by the method of reducing sugars using PAHBAH at pH 4 at various temperatures (see table 1A) or at 60 ° C at different pH (table 1B). Table 1A shows that the temperature optimum of XEA is approximately 80 ° C. Table 1B shows that the optimum pH of XEA is between pH 4 and pH 5 (2 columns of numbers are shown since experiments were performed twice).

Table 1A
Temperature dependence of xylanase Temperature
(° C) Activity (μM xylose / 15 min) Relative
activity (%) thirty 20-30 10 40 40-50 19 fifty 90-100 40 60 120-130 52 70 195-205 83 80 235-245 one hundred 90 65-75 29th

Table 1B
pH dependence pH Activity
(μM xylose / 15 min) Activity
(μM xylose / 15 min) 3.0 118.1 123,4 3,5 141.8 127.8 4.0 145.2 148.3 4,5 140.8 149.1 5,0 117.9 124.8 5.5 91.7 99.1 6.0 57.3 60.1

Thermal stability

Differential Scanning Calorimetric (DSC) scan temperature analysis

The XEA sweep temperature was determined using DSC. The measurement conditions used were with sodium acetate (pH 5), 2-4 mg / ml and a heating rate of 2.5 ° C / min. Found that the deployment temperature (Td) of XEA is 80.1 ° C.

T50 measurement

T50 is the temperature at which 50% residual activity remains after 20 minutes of incubation and, thus, this is a measurement of thermal stability. T50 incubations were performed as follows. The xylanase sample was diluted so that the final xylanase concentration was within the measurement range for the PAHBAH test (XPU units). The sample was then diluted with 0.1 M acetate-sodium buffer (pH 5.0) containing 1 mg / ml BSA to avoid nonspecific binding and denaturation on the surface of the tubes. The buffer was preheated at 60, 70, 80, 85, and 90 ° C in thermal mixers for 5 minutes and then xylanase was added. Samples were heated for 20 minutes and cooled on ice. Activity was measured using the PAHBAH test.

Table 2 shows the percentage of residual activity relative to unincubated control after 20 minutes of incubation at this temperature. T50 can be obtained from this table: it was 82 ° C.

table 2
Thermostability of XEA Xylanase Temperature (° C) Residual Activity (%) 40 one hundred fifty 104 60 103 70 99 75 90 80 85 85 8 90 one

In addition, T50 values were measured at various pH and in the presence of EDTA. The results are presented in table 3.

Table 3
Establishing the effect of pH of metal ions on XEA stability pH T50 (° C) 3 73 3,5 77 four 80 4,5 80 5 81 5 (+ EDTA) 81 6 75 7 ¹ <72 8 ¹ <72 ¹ pH 7 and 8: Too few low-temperature data processing results

XEA is the most stable in the pH range from pH 4 to pH 5. Below pH 3.5 and above pH 5.5, thermal stability begins to deteriorate, but remains much better than most currently known fungal xylanases. The presence of EDTA does not affect the T50. This means that the stability of XEA is independent of the positive metal ions that are complexed by EDTA.

EXAMPLE 7: Use of Talaromyces Xylanase (XEA) in Animal Feeding

The test was performed using male broilers (Cobb). From 1 day to 5 days of age, they were kept in small pens and offered commercial feed for the broiler starter. At the age of 5 days, animals were randomly assigned to 54 cells based on their individual weight. 15 broilers from 5-19 days of age were placed in one cage. On day 19, the number of animals in one cage was reduced to 12. Six cells were allocated to one mode. From this moment on, the cell is an experimental unit, which means that there were 6 repetitions per mode.

Cells were set up in an artificially heated, lit and ventilated broiler house using a three-link cell system. Each cell had an area of 0.98 m ² , wire floor. The room was lit 24 hours / day, but the light intensity was gradually reduced during the test. The temperature was also gradually reduced: from 28 ° C during the first week to 23 ° C during the last week. Humidity during the study was maintained at approximately 60%. Animals were vaccinated according to the routine vaccination program against infectious bronchitis and New Castle disease.

The test included nine modes. No enzyme was added to the wheat-based feed (see table 4) (control), or an amount of approximately 2500, 5000, 10000 or 25000 EXU / kg of feed or Talaromyces endoxylanase (XEA) or Aspergillus niger endoxylanase (EndoI) was added. , endo-1,4-β-endoxylanase ³¹ , commercially available from DSM NV, Agri Ingredients, Delft, Netherlands) as a control. Enzymes were added to the feed in the form of a granular product, which was mixed into a pre-prepared mixture until it was mixed with the feed. Feed was offered to animals ad libitum in the form of balls. During the manufacture of the balls, the temperature of the balls did not exceed approximately 70 ° C. Water was also not limited.

The increase in body weight (PMT) and the coefficient of feed processing (CPC) were determined during the period from 5-19 days of age to 5-33 days of age.

Table 4
Feed composition and essential nutrient content Ingredient Content (%) Wheat 50,0 Rye 10.0 Soya flour 20,0 Fat-free soybeans (dried) 1,5 Cassava 1,69 Meat and bone meal 5.5 Fish flour 2.0 Mixed animal fat 6.0 Mineral and Vitamin Blend ^* 1,0 Limestone 0.85 Calcium monophosphate 0.75 Salt 0.3 L-lysine.HCl 0.16 DL-methionine 0.22 L-threonine 0,03 ME _broilers (MJ / kg) 12.0 Crude protein (%) 21,4 Crude fat (%) 8.5 Easily digestible lysine (%) 1.06 Easily digestible methionine + cysteine (%) 0.78 ^{* The} food contained levels of vitamins and trace minerals, as is customary in the Netherlands. Neither antibiotics nor coccidiostatics were added to the food.

The results of this test are presented in table 5, which shows the average PMT and PDA of broilers in two periods. Enzyme supplements are shown in active units (EXU). As the PMT increases, the CPC decreases, since the CPC is the amount of feed (g) needed for growth.

Table 5 PMT (g / bird) PDA (g / g) 5-19 days 5-33 days 5-19 days 5-33 days The control 647 1665 1,486 1,749 Talaromyces (XEA, according to the invention) +2500 EXU / kg 668 1696 1,447 1,666 +5000 EXU / kg 683 1757 1,419 1,647 +10000 EXU / kg 662 1736 1,444 1,673 +25000 EXU / kg 676 1731 1,429 1,656 Aspergillus niger EndoI (for comparison) +2500 EXU / kg 682 1742 1,440 1,659 +5000 EXU / kg 682 1730 1,458 1,704 +10000 EXU / kg 672 1686 1,417 1,662 +25000 EXU / kg 696 1743 1,466 1,685

In both cases, the growth and CPC significantly improved (P <0.05) for feed containing enzymes, in both cases after 14 days of the experimental period, as well as after the entire experimental period. There were slight differences between the different doses, but the XEA enzyme was such a good (if not better than) commercially available enzyme.

EXAMPLE 8: Bakery Characterization of Talaromyces emersonii Endoxylanase (XEA)

Molded bread was prepared in a standard baking process by mixing 3500 g of wheat flour (a mixture of wheat flour 80% Hummingbird and 20% Ibis (Meneba, Holland) at about 21 ° C), 77 g of pressed yeast (Konings), 70 g of salt, 25 ppm ascorbic acid, 10 ppm Fermizyme ^™ P ₂₀₀ fungal α-amylase, (DSM NV, Bakery Ingredients, Delft, Netherlands) and various amounts of endoxylanase XEA enzyme and 203 ml of water (8-15 ° C) in a spiral mixer (Hobart) for 2 minutes (at 1 speed) and for 6 minutes (at 2 speeds) with an energy consumption of 125 Wh (Watt-hours). The test temperature was 28 ° C. The suitability of the test for processing by machine was determined manually by a qualified baker.

Immediately after mixing, the dough was divided into 6 pieces, 875 g each, rounded and allowed to come up for 35 minutes in a specialized cabinet at 34 ° C and 85% RH (relative humidity). At the end of this period, the test was shaped and granulated and finally allowed to come up 75 minutes in a specialized cabinet at 38 ° C and 87% RH. Subsequently, the fully matched dough was baked in an electric stove at 210 ° C for 30 minutes. After cooling to room temperature, the volume of loaves of bread was determined by the rape seed displacement method. After 16-24 hours of storage in sealed plastic bags at room temperature, the quality of bread crumb was evaluated by a qualified baker. The results are shown in table 6.

Table 6 Dose
XEA
(EXU / kg flour) Volume
loaf
(ml) (%) Butchering
test Bakery
characteristics
(scale 0-10) Quality
crumb
(scale 0-10) 0 4123 one hundred Easily,
not sticky 6 6 176 4420 107 Easily,
not sticky 7 7 527 4756 115 Easily,
not sticky 7.5 7 1581 4794 116 Easy little
slightly sticky 7.5 7.5

The quality of the test was good. Only at the highest dose of XEA endoxylanase did you experience slight stickiness when cutting the dough. However, this slight stickiness did not affect the suitability of the dough for machine processing. The whole dough containing endoxylanase XEA was very flexible and easy to handle.

Based on these bakery results, it was concluded that endoxylanase XEA is very effective in improving the quality of bread, both in terms of loaf volume and in the quality of bread crumb. Despite the large volumes of loaves, the structure of the bread crumb remained uniform and of high quality.

EXAMPLE 9 and COMPARATIVE EXAMPLE 10: Comparison of the baking characteristics of Talaromyces emersonii enzyme (XEA) with endoxylanase from Asp.niger

The baking performance of XEA was compared in the production of Dutch molded bread to the currently used mushroom endoxylanase from Aspergillus niger. This A.niger endoxylanase was supplied in pure commercially available form, i.e. Fermizyme ^TM HSP ₆₀₀₀ . Fermizyme ^TM HSP is a good enzyme for use in making bread, but it can lead to sticky dough and does not always provide a significant increase in loaf volume.

The process of Example 7 was repeated exactly, except that various amounts of either XEA endoxylanase or Fermizyme ^TM HSP _{6000 were used.}

The results are shown in table 7.

Table 7 Example No. Dose
(EXU / kg
flour) Loaf volume
(ml) (%) Butchering
test Brittleness and
crumbling
(scale 0-10) Quality
crumb (0-10 scale) 9:
endoxylanase
XEA 0 4170 one hundred Good not sticky 6 6 264 4430 106 Good
not sticky 6.5 7.5 528 4647 111 Good
not sticky 7 7.5 1056 4761 114 Elastic
not sticky 7.5 8 1584 4880 117 Stretchy, slightly baked, non-sticky 5# 7.5 10:
Fermizyme ^TM
HSP ₆₀₀₀ 0 4170 one hundred Good
not sticky 6 6 264 4304 103 Elastic
not sticky 6.5 6 528 4355 104 Elastic
Little
sticky 6.5 7 1056 4539 109 Elastic
sticky
Little
unbaked 7 7.5 1584 4698 113 Elastic
sticky
Little
unbaked 7.5 8 # the volume of the loaf was too large, so the bread was shaped like a mushroom.

From the results obtained, it is clear that XEA endoxylanase does not lead to stickiness of the test and the loaf volume is increased to a greater extent than that obtained by the introduction of Fermizyme ^TM HSP. Moreover, for a typical loaf volume, flour with lower units / kg of endoxylanase was required when using XEA instead of Fermizyme ™ HSP. The brittleness and quality of cutting were similar at equal volumes. The crumb structure obtained by the addition of XEA was at least as good as that obtained using Fermizyme ^TM HSP.

In general, XEA endoxylanase solves some of the problems associated with dough and bread making using Fermizyme ^TM HSP. The dough did not appear sticky, the volumes of loaves obtained were larger and the structure of the bread crumb remained uniform and of high quality despite the larger volumes.

EXAMPLE 11: Testing the stability of the granulation

Corn starch (4543 g) was placed in an Erweka ^TM Z-mixer. Then, 1069 g of the ultrafiltrate from the fermenter containing xylanase according to this invention (a portion of XEA 502-8m containing 43553 EXU / g and 6.7% dry matter) was added to starch with stirring until a crude mixture containing about 15000 EXU / g (at drying to 94% dry matter). After adding the liquid, the mixture was left for another 10 minutes.

In a vacuum dryer (40 ° C), the mixture was dried for 12 hours to obtain 94.5% dry matter. It was then ground in an Erweka ^TM Freewitt mill through a 1 mm grate. This was an example of 11 A.

The same method was repeated using Lyxasan ^™ Batch OP 0036 (containing endo-1,4-β-endoxylanase ³¹ , commercially available from DSM NV, Agri Ingredients, Delft, Netherlands). To 5885 g of starch was added 1984 g of UV (ultrafiltrate) with 42550 EXU / g and 3.7% dry matter. This mixture was dried to 94% dry matter and crushed, as in Example 11A (this constitutes Comparative Example 11B).

The third sample is a commercially available product called Biofeed Wheat CT, which is commercially available from Novo Nordisk, Denmark. It contains G-type xylanase from Thermomyces lanuginosis and has a fat coating (comparative example 11C).

All three samples were tested in a granulation study at three different temperatures. An enzyme mixture was added to the animal feed (composition shown in table 8 below) in two different concentrations, 0.24% (11A) and 0.1% (11B, 11C). After mixing, the feed was heated with steam in an air conditioner to 65 ° C, 75 ° C or 85 ° C and subsequently granulated through a 65 mm sieve with holes 5 mm in diameter. Chilled immediately. Then, the residual activity in the beads was measured, and the results of the stability tests are shown in Table 9.

Table 8
The composition of animal feed Raw materials Content (%) Corn 20.00 Wheat 30.00 Soybeans (heat treated) 10.00 Soybeans (flour) (46.7 cp) 22.50 Tapioca 5.07 Fishmeal (70% cp) 1,50 Feather meal (hydrolyzed) 1.00 Soybean oil 1.30 Animal fat 4,50 Vitamin / Mineral Mixture (Corn) 1.00 Lemeston 1,300 Calcium monophosphate 1.20 Salt 0.32 L-lysine 0.12 DL-methionine 0.19

Table 9
The stability of enzymes during granulation Residual Example 11A Comparative Comparative activity(%) example 11B example 11C 65 ° C 84 64 86 75 ° C 82 16 83 85 ° C 66 four 56

As can be seen, the stability at high temperature (85 ° C) of the XEA protein according to this invention showed better results than the currently sold commercial product.

EXAMPLE 12: Activity against aryl-β-D-xylosides

Two substrates 5-bromo-4-chloro-3-indoxyl-β-D-xylopyranoside (CAS Reg No.207606-55-1, also called XbD-xyl) and 4-umbelliferyl-β-D-xylopyranoside (CAS Reg No .6734-33-4, also called 4-MU-bD-xyl) was used to screen for xylosidase activity. They were added directly to the medium. The strains making up the library were located on a 96-well plate. In this way, the control plate was easily replicated on the determination medium. The library was grown on selective medium (0.52 g / L KCl, 1.52 g / L K ₂ HPO ₄ , 0.52 g / L MgSO ₄ , 2% glucose, 10 mM acetamide, 1/1000 trace elements (2.2 g ZnSO ₄ / 7H ₂ O, 1.1 g H ₃ BO ₃ , 0.5 g FeSO ₄ / 7H ₂ O, 0.17 g CoCl ₂ / 6H ₂ O, 0.16 g CuSO ₄ / 5H ₂ O, 0.15 g NaMoO ₄ / 2H ₂ O, 5.0 g EDTA, pH adjusted to 6.5 with KOH and a sterilized 0.45 μm filter), pH adjusted using KOH (10 N to 6) containing either XbD -xyl or 4-MU-bD-xyl (at 200 mg / L and 150 mg / L, respectively). XbD-xyl was pre-dissolved in a minimum volume of dimethylformamide (DMF). The plates were incubated at 33 ° C for 48 hours and then incubated for 6 hours n at 65 ° C. The plates were evaluated before and after 6-hour incubation at 65 ° C. X-xyl plates were analyzed immediately based on the presence (or absence) of a turquoise blue zone, the presence of which was detected. Determination of xylosidase activity on 4-MU-xyl the tablets were checked by installing the tablet under ultraviolet light with a wavelength of 310 nm. Positive clones appeared surrounded by a blue fluorescent zone.

Claims (23)

1. The polypeptide having xylanase activity, containing (i) amino acid sequence 23 to 408 of amino acid SEQ ID No. 2; or (ii) option (i) having the ability to cleave xylan, while at least 70% it is identical to the amino acid sequence from 23 to 408 amino acid SEQ ID No. 2; or (iii) a fragment of (i) or (ii) having the ability to cleave xylan. 2. The polypeptide according to claim 1, in which option (ii) is at least 80% identical to the amino acid sequence from 23 to 408 amino acid SEQ ID No. 2 and / or a fragment (iii) of at least 150 amino acids in length. 3. The polypeptide according to claim 1 or 2, cleaving (1 → 4) bonds or adjacent xylopyranosyl units in β-D-xylan. 4. The polypeptide according to one of claims 1 to 3, having arabinoxylase and xylosidase activity. 5. The polypeptide according to one of claims 1 to 4, obtained from a fungus or organism of the genus Talaromyces, for example, the species Talaromyces emersonii. 6. Polynucleotide encoding a polypeptide having xylanase activity, containing (a) the nucleotide sequence of SEQ ID No.1 or a sequence encoding a polypeptide according to one of claims 1 to 4; (b) a sequence complementary to the sequence or hybridizing under stringent conditions with the sequence of subparagraph (a); (c) a fragment with a length of at least 100 nucleotides of the sequence according to subparagraphs (a) or (b); (d) a sequence that is at least 70% identical to the sequence of subparagraphs (a), (b) or (c); or (e) a sequence genetically degenerate with respect to one of the sequences of subparagraphs (a) to (d). 7. The polynucleotide of claim 6, wherein the fragment of subparagraph (c) has a length of at least 200 bases and / or the identity of subparagraph (d) is at least 80%. 8. The polynucleotide according to claim 7, containing (a) a sequence encoding a polypeptide having xylanase activity, which is: (1) the coding sequence of nucleotides 69 to 1224 of the sequence of SEQ ID No.1; (2) a sequence that, under stringent conditions, hybridizes with the complementary sequence of subparagraph (1); or (3) a sequence genetically degenerate with respect to the sequence of subparagraphs (1) or (2); or (b) a sequence complementary to the polynucleotide of subparagraph (a). 9. The polynucleotide according to one of claims 6 to 8, which is a DNA sequence. 10. An expression vector containing a polynucleotide according to one of claims 6 to 9. 11. The vector of claim 10, operatively associated with a regulatory sequence. 12. A method for producing a polypeptide according to one of claims 1 to 5, comprising culturing a host cell containing, as a heterologous sequence, a polynucleotide according to one of claims 6 to 9, under conditions ensuring expression of the polypeptide. 13. The method according to item 12, in which the host cell is a host cell expressing as a heterologous protein a polypeptide according to one of claims 1 to 5. 14. The method of claim 12, wherein the host cell is a host cell transformed with the DNA sequence of claim 9 or the vector of claim 10. 15. A composition having xylanase activity, containing the active substance and conventional carriers or diluents, characterized in that it contains the polypeptide according to one of claims 1 to 5 as an active substance. 16. The composition according to p. 15, characterized in that it further comprises a polypeptide having cellulase, endoarabinanase, ramogalacturonase or polygalacturonase activity. 17. A method of processing a plant or xylan-containing material, comprising contacting said material with a polypeptide according to one of claims 1 to 5 or a composition according to claim 15 or 16. 18. The method according to 17, in which the contacting is directed to the decomposition, hydrolysis or modification of xylan in the specified material or the destruction or modification of the cell walls of plant material. 19. The method according to 17, in which the contacting is directed to the cleavage of xylanopyranosyl or β-D-xylan subunits of the specified material. 20. The method according to 17, in which the contacting is aimed at reducing the viscosity or improving the transparency or filterability of the specified material. 21. The processed material obtained by contacting a plant or xylan-containing material with a polypeptide according to one of claims 1 to 5, or a composition according to claim 15 or 16. 22. Animal feed containing ingredients from the group of proteins, carbohydrates, fats, vitamins, trace elements and mixtures thereof, as well as the polypeptide according to one of claims 1 to 5. 23. Bread containing a polypeptide according to one of claims 1 to 5.