Xylanase mutants

Abstract

Disclosed are mutant polypeptides of xylanase, as well as fusion proteins and enzyme compositions comprising the mutant polypeptides, recombinant host cells for the production of the mutant polypeptides, methods for and uses thereof, wherein the mutant polypeptide comprises an amino acid sequence having 79% or more but less than 100% amino acid sequence identity with amino acids 1-191 of SEQ ID NO:1, the amino acid sequence having xylanase activity, at least one disulfide bridge between two Cys residues within the amino acid region of 1-191, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

Description

[Technical field]

[0001] The present disclosure relates generally to the field of enzyme technology. The present disclosure particularly, but not exclusively, relates to xylanase variants that exhibit improved xylan degradation ability and stability in various chemical and thermal environments, making them useful in a variety of applications where degradation of xylan is desired, such as pulp or silage processing, and feed or food production. [Background technology]

[0002] This section provides useful background information without any admission that any of the techniques described herein represent the state of the art.

[0003] Plant cell walls consist primarily of a complex mixture of polysaccharides, mainly cellulose, lignin and hemicellulose. Xylan is the major hemicellulose component in most materials derived from plants and consists of a polysaccharide backbone of β-1,4 linked xylose residues, often with acetyl, arabinosyl and glucuronosyl substituents. Endo-β-1,4-xylanase (EC 3.2.1.8) is an enzyme that acts on xylans and xylo-oligosaccharides by degrading linear polysaccharides, resulting in a mixture of smaller xylooligosaccharides and xylose (monosaccharides).

[0004] The demand for industrially applicable xylanases that hydrolyze xylan has increased significantly, and there is a continuous need for enzymes with improved properties. Xylanases that are highly active and stable at high temperatures as well as at alkaline pH are particularly desirable in many industrial processes, such as the enzyme-catalyzed bleaching of pulp, where xylanases are used as a simple and cost-effective alternative to toxic chlorine-containing chemicals.

[0005] Xylanases are also useful as additives to animal feeds, as they have beneficial effects in improving feed nutrient utilization. Plant-based animal feeds contain xylan, but in large quantities xylan can impair nutrient digestion, microbial colonization and livestock growth. Addition of xylanase enzymes to animal feeds has beneficial effects such as increased livestock growth rate, increased feed conversion and increased feed efficiency. Solubilization and degradation of xylan by xylanase reduces viscosity in the livestock intestine and increases the release of nutrients from the endosperm and aleurone layers of cereal grains. It has also been suggested that xylooligosaccharides (XOS), sugar oligomers composed of xylose units, act as prebiotics and confer various health benefits to livestock animals.

[0006] Other industrial or commercial applications in which xylanases are utilized are, for example, silage processing, biomass conversion for biofuel production, and the starch, food, baking (e.g., bread making) and beverage (e.g., fruit juice and wine clarification, or alcoholic fermentation) industries.

[0007] Actinomycetes are known to produce xylanases, some of which produce highly thermostable and alkali-tolerant xylanase enzymes. Thermophilic actinomycete strains are useful sources of xylanases for industrial processes. Xylanase polypeptides contain a catalytic module (core) and many also contain a carbohydrate-binding module (CBM) separated from the core by a linker region. Xylanolytic enzymes with the above-defined modular structure are produced by fungi and bacteria, as well as by strains of actinomycetes including Actinomadura (Nonomuraea) flexuosa (e.g., N. flexuosa Xyn11A and Xyn10A consist of a core, linker and CBM).

[0008] The present disclosure describes mutants of actinomycete xylanases and mutants of truncated Actinomadura (Nonomuraea) flexuosa Xyn11 xylanase named AM24 (Nf Xyn11A, also named AM35). AM24 xylanase and methods for its production were described in US Patent No. 5,399,663. AM24 is a truncated xylanase of full-length xylanase AM35 from Actinomadura (Nonomuraea) flexuosa. AM24 differs from full-length xylanase AM35 in that the carbohydrate binding domain (CBM) and part of the linker between the core and CBM have been genetically removed. Full-length Actinomadura (Nonomuraea) flexuosa Xyn11A (Nf Xyn11A, AM35) xylanase protein has also been previously described (US Patent No. 5,399,663). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] European Patent No. 1737951 Summary of the Invention

[0010] It is an object of the present disclosure to provide xylanase variants that exhibit xylanase activity, and have performance and stability that allows for their use in industrial processes. Another object of the present disclosure is to provide xylanase variants that have improved properties when used in industrial processes. Yet another object of the present disclosure is to provide variants that can be used in enzyme compositions for the degradation or modification of xylans.

[0011] The present application relates to the invention as defined in the accompanying independent claims and to the embodiments thereof disclosed below.

[0012] The present invention is based on the development of xylanase variants by modifying the parent xylanase polypeptide AM24 or another actinomycete xylanase polypeptide homologous to AM24. The inventors have surprisingly found that by incorporating at least one disulfide bridge and an amino acid substitution at the position corresponding to position 23 or 28 or both positions 23 and 28 of the parent xylanase variant polypeptide, it is possible to obtain variants with improved, i.e. enhanced, performance of xylan degradation, as well as improved stability, compared to the parent xylanase AM24 or parent AM24 xylanase homolog, as shown in the following examples. The performance and stability are improved, especially when the reaction conditions require high temperatures. The novel variant polypeptides of xylanase have high performance and stability in a wide range of industrial processes, thus providing improved properties for enzyme compositions for the degradation and modification of xylans in those processes.

[0013] According to a first aspect, there is provided a mutant polypeptide comprising an amino acid sequence having an amino acid sequence identity of 79% or more but less than 100% with amino acids 1 to 191 of SEQ ID NO:1, the amino acid sequence being Xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region; and Amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1; A mutant polypeptide is provided having the following structure:

[0014] The present variant polypeptides are advantageous in that they have improved thermostability, pH stability and performance, i.e. xylanolytic activity, and these characteristics can be measured as described in Examples 4 and 5.

[0015] In certain embodiments, functional fragments of the present variant polypeptides of xylanase are provided.

[0016] According to a second aspect, there is provided a method for the preparation of a polypeptide comprising the amino acid sequence of the variant of the first aspect, an amino acid sequence providing a secretory signal sequence; an amino acid sequence that facilitates purification, e.g., an affinity tag or a His tag; Amino acid sequences that control and enhance the production of recombinant polypeptides, e.g., carrier polypeptides; an amino acid sequence encoding a protease cleavage site; an amino acid sequence having enzymatic activity; and / or An amino acid sequence that provides the fusion protein with binding affinity, e.g., a carbohydrate-binding moiety. At least one of A fusion protein is provided, comprising:

[0017] According to a third aspect there is provided an enzyme composition comprising the variant polypeptide of the first aspect or the fusion protein of the second aspect.

[0018] The enzyme compositions are particularly effective in such processes because the xylanase variants have good stability and specific activity when used to degrade xylan in processes such as pulp processing, such as enhancing the pulp bleaching process and improving refining efficiency, as well as industrial feed or food production.

[0019] According to a fourth aspect, there is provided a recombinant host cell comprising genetic elements which enable it to produce at least one recombinant polypeptide comprising the mutant polypeptide of the first aspect or the fusion protein of the second aspect.

[0020] The variant polypeptides of the present disclosure have properties that make them useful in enzyme compositions for industrial applications.

[0021] In one embodiment, an enzyme preparation comprising a mutant polypeptide having xylanase activity and obtainable by using the host cell is provided.

[0022] According to a fifth aspect, there is provided a method for producing a variant polypeptide of the first aspect or a fusion protein of the second aspect, the method comprising: a. culturing a recombinant host cell of the fourth aspect under conditions that allow the production of the recombinant polypeptide; and optionally b. Recovering the recombinant polypeptide. Includes.

[0023] The above method provides an efficient way to generate a xylanase variant polypeptide or a fusion protein comprising a xylanase variant polypeptide and at least one amino acid sequence according to the second aspect. The variant polypeptide is produced in a recombinant host cell, providing a production system that can be optimized, tuned and controlled in a desired manner. The variant polypeptides produced by the method differ from the parent xylanase at the structural and functional levels.

[0024] According to a sixth aspect there is provided a method for degrading or modifying xylan-containing material, the method comprising treating said xylan-containing material with an effective amount of a mutant polypeptide of the first aspect, a fusion protein of the second aspect or an enzyme composition of the third aspect. In an embodiment the treatment comprises contacting the xylan-containing material with the mutant polypeptide, fusion protein or enzyme composition in an aqueous medium.

[0025] According to a seventh aspect there is provided the use of the variant polypeptide of the first aspect or the fusion protein of the second aspect or the enzyme composition of the third aspect in an industrial process or commercial application such as pulp or silage processing.

[0026] According to an eighth aspect there is provided the use of the variant polypeptide of the first aspect or the fusion protein of the second aspect or the enzyme composition of the third aspect in the preparation of a feed or foodstuff.

[0027] In an embodiment there is provided the use of the variant polypeptide of the first aspect or the fusion protein of the second aspect or the enzyme composition of the third aspect in processing pulp, for example to enhance lignin removal and / or cellulose hydrolysis, and to increase the susceptibility of the pulp to refining.

[0028] In an embodiment there is provided the use of a variant polypeptide of the first aspect or a fusion protein of the second aspect or an enzyme composition of the third aspect in an animal feed, the feed being used to improve the weight gain and feed efficiency of the animal for feeding the animal, where appropriate.

[0029] In an embodiment there is provided a use of the variant polypeptide of the first aspect or the fusion protein of the second aspect or the enzyme composition of the third aspect in the preparation of a foodstuff. The variant polypeptide may be used in coffee extraction, vegetable oil or starch processing, removal of gums from vegetable fibre sources, baking, dough conditioning, bread making and the beverage industry (e.g. clarification or alcoholic fermentation of fruit juice and wine).

[0030] The present xylanase variants are advantageous in that they have improved performance in degrading xylan in process conditions requiring increased stability. As shown in the examples provided below, the claimed xylanase variant polypeptides have improved performance and stability when compared to the parent AM24 xylanase or parent AM24 xylanase homologs, depending on the origin of the variant.

[0031] Various non-binding exemplary aspects and embodiments have been described above. The foregoing embodiments are used only to illustrate selected aspects or steps that may be utilized in various embodiments. Some embodiments may be presented merely with reference to certain exemplary aspects. It should be recognized that the corresponding embodiments may also be applied to other exemplary aspects.

[0032] Several illustrative embodiments will now be described with reference to the accompanying drawings. [Brief description of the drawings]

[0033] [Figure 1A] Figure 1A shows the phylogenetic tree of the AM24 core sequence and its homologues. The tree is divided into two parts, Figure 1A and 1B, with arrows representing the connections between the two figures. A phylogenetic tree from the 100 closest homologues to the AM24 core, designed using the blastp search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) and the phylogenetic tree option of the Clustal Omega multiple sequence alignment program (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ). The amino acid sequence of AM24 (Nf_Xyn11A core, amino acids 1-191 of SEQ ID NO: 1, the core region according to Leskinen et al., 2005) was used as the query sequence. The algorithm parameters used are explained in more detail in Example 2. [Figure 1B] Figure 1B shows the phylogenetic tree of the AM24 core sequence and its homologues. The phylogenetic tree is divided into two parts, Figure 1A and 1B, with arrows representing the connections between the two figures. A phylogenetic tree from the 100 closest homologues to the AM24 core, designed using the blastp search (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins) and the phylogenetic tree option of the Clustal Omega multiple sequence alignment program (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ). The amino acid sequence of AM24 (Nf_Xyn11A core, amino acids 1-191 of SEQ ID NO: 1, the core region according to Leskinen et al., 2005) was used as the query sequence. The algorithm parameters used are described in more detail in Example 2. [Diagram 2]Figure 2 shows an alignment of the amino acid sequence of AM24 with the mature putative core regions of close homologs selected for production in Trichoderma reesei. The alignment of these sequences was performed using Clustal Omega (1.2.4) multiple sequence alignment. The mature core region of AM24 consists of amino acids 1-191 of SEQ ID NO: 1, based on information published in Leskinen et al. (2005). The alignment in Figure 2 displays the following symbols representing the degree of conservation observed in each column: * (asterisk) indicates a position with a single completely conserved residue; : (colon) indicates a position with conservation between groups with very similar properties; . (period) indicates a position with conservation between groups with weakly similar properties. [Figure 3A] Figure 3A shows the relative thermostability of AM24 and its mutants at pH 7. Xylanase activity analysis was performed at temperatures of 70, 80, 90 and 96°C using reaction times of 5 and 60 min. Figure 3A: Results of the 5 min reaction time assay. Activity values ​​were relative to the activity of the corresponding xylanase analyzed at 80°C using a reaction time of 5 min. This activity was set to 100. Values ​​presented are the average from two parallel reactions. AM24 represents the parent xylanase, the other samples represent different AM24 mutants. [Figure 3B] Figure 3B shows the relative thermostability of AM24 and its mutants at pH 7. Xylanase activity analysis was performed at temperatures of 70, 80, 90 and 96°C using reaction times of 5 and 60 min. Figure 3B: Results of the 60 min reaction time assay. Activity values ​​were relative to the activity of the corresponding xylanase analyzed at 80°C using a reaction time of 5 min. This activity was set to 100. Values ​​presented are the average from two parallel reactions. AM24 represents the parent xylanase, the other samples represent different AM24 mutants. [Figure 4A]Figure 4A shows the relative thermostability of AM24 and its mutant C31-4 at pH 8 and 9. Xylanase activity analysis was performed at 70, 80, 90 and 96°C with a reaction time of 5 min, and at 70 and 80°C with a reaction time of 60 min. Figure 4A: Reaction conditions pH 8, 5 min reaction. Activity values ​​were for the corresponding xylanase activity analyzed at pH 7, 80°C with a reaction time of 5 min. This value was set to 100. These values ​​are the average from two parallel reactions. [Figure 4B] Figure 4B shows the relative thermostability of AM24 and its mutant C31-4 at pH 8 and 9. Xylanase activity analysis was performed at 70, 80, 90 and 96°C with a reaction time of 5 min, and at 70 and 80°C with a reaction time of 60 min. Figure 4B: Reaction conditions pH 8, 60 min reaction. Activity values ​​were relative to the activity of the corresponding xylanase analyzed at pH 7, 80°C with a reaction time of 5 min. This value was set to 100. These values ​​are the average from two parallel reactions. [Figure 4C] Figure 4C shows the relative thermostability of AM24 and its mutant C31-4 at pH 8 and 9. Xylanase activity analysis was performed at 70, 80, 90 and 96°C with a reaction time of 5 min, and at 70 and 80°C with a reaction time of 60 min. Figure 4C: Reaction conditions pH 9, 5 min reaction. Activity values ​​were relative to the activity of the corresponding xylanase analyzed at pH 7, 80°C with a reaction time of 5 min. This value was set to 100. These values ​​are the average from two parallel reactions. [Figure 4D] Figure 4D shows the relative thermostability of AM24 and its mutant C31-4 at pH 8 and 9. Xylanase activity analysis was performed at 70, 80, 90 and 96°C with a reaction time of 5 min, and at 70 and 80°C with a reaction time of 60 min. Figure 4D: Reaction conditions pH 9, 60 min reaction. Activity values ​​were relative to the activity of the corresponding xylanase analyzed at pH 7, 80°C with a reaction time of 5 min. This value was set to 100. These values ​​are the average from two parallel reactions. [Diagram 5]Residual xylanase activity of AM24 and its mutant C31-4 after incubation at pH 11, 90°C for 60 min. Residual xylanase activity as a percentage of the original activity in the samples was analyzed at pH 7, 70°C with a reaction time of 5 min. Results are from two different analyses, two parallel reactions in each. BR, Britton-Robinson buffer; NaP, sodium phosphate buffer. Xylanase activity analyzed before incubation at pH 7, 70°C with a reaction time of 5 min was set to 100.

[0034] Sequence Listing TIFF2024501524000002.tif234155 TIFF2024501524000003.tif245155 TIFF2024501524000004.tif220155 TIFF2024501524000005.tif225155 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention relates to mutant xylanase polypeptides having substitutions introduced into the amino acid sequence that result in improved properties, such as improved thermostability and pH stability.

[0036] As used herein, the term "homolog" or "xylanase homolog" refers to a DNA, RNA or amino acid xylanase sequence that has a certain level of biological homology, i.e., similarity to another DNA, RNA or amino acid xylanase sequence, expressed as the % of residues that match the sequence or as the % of sequence identity / similarity. In one embodiment, the xylanase homolog is an AM24 xylanase homolog.

[0037] The term "xylan" refers to a matrix polysaccharide or heteropolymer composed of a backbone of repeating β-1,4 linked xylose residues with various side chains. Xylan polysaccharides can be broadly divided into the classes O-acetylglucuronoxylan (AcGX), O-acetylarabinoxylan (AcAX), O-acetylglucuronoarabinoxylan (AcGAX) and arabinoglucuronoxylan (AGX) based on the substituted side chains. Xylan-containing materials include plant-based or plant-derived materials.

[0038] As used herein, the term "xylanase" refers to a xylanase enzyme defined according to what is known in the art as endo-1,4-β-xylanase or 4-β-D-xylan xylanohydrolase, which is known to catalyze the endohydrolysis of (1→4)-β-D-xylosidic bonds in xylan. Xylan has the alternative names endo-(1→4)-β-xylan 4-xylanohydrolase, endo-1,4-xylanase, xylanase, β-1,4-xylanase, endo-1,4-xylanase, endo-β-1,4-xylanase, endo-1,4-β-D-xylanase, 1,4-β-xylan xylanohydrolase, β-xylanase, β-1,4-xylan xylanohydrolase, endo-1,4-β-xylanase and β-D-xylanase. Xylanases are classified as EC 3.2.1.8 according to the enzyme nomenclature.

[0039] As used herein, Nf xyn11A refers to the Nonomuraea flexuosa xylanase gene that encodes the family 11 xylanase Nf Xyn11A, also designated AM35.

[0040] As used herein, AM35 refers to the wild-type mature xylanase that is the parent polypeptide used in the AM24 polypeptide. The amino acid of the mature AM35 protein corresponds to the amino acid sequence of SEQ ID NO:97.

[0041] As used herein, AM24 (also referred to herein as AM24 protein) is the parent polypeptide of the AM24 mutant polypeptides of the present disclosure. AM24 is encoded by a truncated DNA sequence that encodes a truncated form of xylanase AM35 (Nf Xyn11A). The AM24 protein contains the catalytic module (core) and lacks the carbohydrate binding module (CBM) and a portion of the linker region between the core and CBM. The amino acids of the mature polypeptide of AM24 correspond to the amino acid sequence of SEQ ID NO:1, including amino acids D1 to L220, whereas the "mature core polypeptide" of AM24 includes amino acids D1 to G191 of SEQ ID NO:1.

[0042] As used herein, the term "variant" refers to a sequence or a fragment of a sequence (nucleotides or amino acids) that has been inserted, substituted or deleted by one or more nucleotides / amino acids, or that has been chemically modified. The term variant may also include, in some embodiments, a variant polypeptide of xylanase, a fusion protein comprising a variant polypeptide of xylanase, or a recombinant xylanase enzyme.

[0043] The terms "xylanase variant" and "variant xylanase" and "variant of xylanase" refer to any xylanase molecule obtained by site-directed or random mutagenesis, insertion, substitution, deletion, recombination and / or any other protein engineering method that results in a xylanase that differs in amino acid sequence from a parent xylanase, which parent xylanase being a wild-type xylanase or a xylanase variant itself. The terms "wild-type xylanase", "wild-type enzyme", "wild-type" or "wt" according to the present disclosure describe a xylanase enzyme having an amino acid sequence found in nature or a fragment thereof. A gene encoding a variant can be synthesized or the parent gene can be modified using genetic methods, for example, by site-directed mutagenesis, a technique that introduces one or more mutations at one or more defined sites in a polynucleotide encoding a parent polypeptide.

[0044] The term "mutant polypeptide" of the xylanase according to the first aspect may also be referred to using the name given to the mutant, for example C31-4 or mutant C31-4.

[0045] As used herein, the term "homolog mutant" or "mutant of a homolog" refers to any mutant molecule derived from an AM24 xylanase homolog, obtained by synthesizing the corresponding gene or by modifying the parent gene by site-directed or random mutagenesis, insertion, substitution, deletion, recombination and / or any other protein engineering method that results in an AM24 xylanase homolog mutant that differs in amino acid sequence from the parent xylanase, the parent xylanase homolog being a wild-type xylanase or a xylanase mutant itself. In one embodiment, phrases such as "xylanase homolog mutant of XB1" refer to a mutant of an AM24 xylanase homolog, the homolog being XB1.

[0046] As used herein, the term "mature polypeptide" refers to any polypeptide from which at least one signal sequence or signal peptide or signal peptide and putative propeptide has been excised. For example, the "mature polypeptide" of AM24 comprises amino acids D1 to L220 of SEQ ID NO:1, and the "mature core polypeptide" of AM24 comprises amino acids D1 to G191 of SEQ ID NO:1.

[0047] As used herein, "peptide" and "polypeptide" are amino acid sequences that contain multiple consecutive polymerized amino acid residues. For the purposes of this disclosure, a peptide is a molecule that contains up to 20 amino acid residues, and a polypeptide contains more than 20 amino acid residues. A peptide or polypeptide may contain modified amino acid residues, naturally occurring amino acid residues that are not encoded by codons, and non-naturally occurring amino acid residues. As used herein, "protein" may refer to a peptide or polypeptide of any size. A protein may be an enzyme, a protein, an antibody, a membrane protein, a peptide hormone, a regulator, or any other protein.

[0048] As used herein, "sequence identity" means the percentage of exact matches of amino acid residues between two optimally aligned sequences over the number of positions where the residue occurs in both of the two sequences. When one sequence has a residue that has no corresponding residue in the other sequence, the alignment program allows a gap in the alignment and that position is not counted in the denominator of the identity calculation.

[0049] As used herein, "sequence alignment" of amino acid sequences means aligning sequences using the Clustal Omega (1.2.4) multiple sequence alignment program (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) as described by Sievers et al. 2011, using default settings.

[0050] Unless otherwise specified, all references to a particular amino acid position refer to the amino acid of SEQ ID NO:1 at that position, or the amino acid present or absent at the corresponding position in an amino acid sequence aligned with SEQ ID NO:1.

[0051] As used herein, the term "disulfide bridge" or "disulfide bond" or "SS bridge" refers to a bond formed between the sulfur atoms of cysteine ​​residues in a polypeptide or protein. Disulfide bridges may be naturally occurring or non-naturally occurring and may be introduced, for example, by amino acid substitution.

[0052] As used herein, the term "corresponding position" or "corresponding amino acid position" refers to aligning at least two amino acid sequences according to a specified region that is similar or identical as a pairwise alignment or multiple sequence alignment, thereby pairing corresponding amino acids. An example of a corresponding position is shown in Figure 2, where the amino acids shown in the same column are corresponding amino acids. For example, at position 1, amino acid D1 of AM24 and Q1 of XB1 are corresponding amino acids.

[0053] As used herein, an "amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue that is different from the original amino acid at that particular substitution position. The term "amino acid substitution" can refer to both conservative and non-conservative amino acid substitutions, meaning that an amino acid residue is replaced with an amino acid residue having a similar side chain (conservative) or a different side chain (non-conservative) than the original amino acid residue at that position.

[0054] The term "functional fragment" refers to a fragment or portion of the current variant polypeptide of xylanase that retains substantially the same enzymatic function or effect.

[0055] "Xylanase activity" as used herein refers to xylanolytic activity. Degradation or modification as used herein means that xylan polysaccharides are hydrolyzed by xylanases to xylose oligosaccharides or xylose disaccharides. The xylanolytic activity of the polypeptides according to the present disclosure can be tested according to testing procedures known in the art. Examples 4 and 5 provide examples of methods for measuring xylanase activity. The term "xylanase activity retention" refers to the ability of an enzyme to retain xylanase activity when exposed to a particular process condition. In an embodiment, increased activity retention of a xylanase enzyme means that the xylanase enzyme better retains its xylanase activity in a particular process condition when compared to the parent xylanase AM24 in the same condition.

[0056] In one embodiment, the term "enzyme composition" refers to an enzyme fermentation product, optionally isolated and purified, from a single species of microorganism. An enzyme composition usually contains several different enzymatic activities produced by a microorganism. In another embodiment, the enzyme composition is a mixture of single component enzymes, preferably a mixture of enzymes obtained from bacterial or fungal species using conventional recombinant techniques. The enzymes are obtained from the fermentation of a microorganism and are optionally isolated and purified. The enzymes in the mixture can be from different species, preferably fungal or bacterial species, or are present in the fermentation product of a microorganism acting as a host cell for the production of other enzymes (e.g., for the production of recombinant xylanase) when the microorganism simultaneously produces other enzymes in addition to the recombinant xylanase.

[0057] The term "stability" in the context of enzyme or xylanase stability describes the ability of an enzyme to withstand and / or function under process conditions that are not optimal for the activity and function of the enzyme in question, such as high temperature, high pH or high radiation, certain concentrations of inorganic salts or organic solvents, or particular reaction mixture compositions, including, for example, proteases, stabilizers, builders, surfactants, etc.

[0058] The term "stability" reflects the stability of the xylanases of the present disclosure over time, e.g., how much activity is retained when the xylanase is exposed to process conditions that are not optimal for the activity and function of the enzyme in question. Various methods are used to analyze the stability of xylanase variants. The thermostability of xylanases can be assessed by measuring the unfolding temperature of xylanases, as described in Example 5. Also, the stability and residual activity of xylanases can be measured using "activity assays" with adjusted parameters, as described in Examples 4 and 5. The term "stability" includes stability during use in processes having high temperature conditions.

[0059] As used herein, "host cell" means any cell type that is susceptible to transformation, transfection, transduction, conjugation, crossover, etc. with a nucleic acid construct or expression vector comprising a polynucleotide. The term "host cell" encompasses any progeny that are not identical due to mutations that occur during replication. Non-limiting examples of host cells are fungal cells, preferably filamentous fungal cells (e.g., Trichoderma or Trichoderma reesei, Aspergillus or Aspergillus oryzae or Aspergillus niger, Thermothelomyces or Thermothelomyces heterothallica or Humicola or Humicola insolens or Fusarium or Fusarium venenatum), bacterial cells, preferably gram-positive bacilli (e.g., Bacillus subtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens, B. pumilus), gram-negative bacteria (e.g., Escherichia coli), actinomycetes (e.g., Streptomyces sp., Nonomuraea flexuosa) and yeasts (e.g., Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica).

[0060] The term "promoter" refers to a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding region of a gene.

[0061] The term "secretory signal sequence" or "signal sequence" or "secretory peptide" refers to an amino acid sequence that is a component or part of a larger polypeptide that directs the larger polypeptide through the secretory pathway of the host cell in which it is produced. Secretory signal sequences can be natural or can be obtained from another source. Depending on the host cell, the larger polypeptide can be cleaved from the secretory peptide during passage through the secretory pathway, thereby forming a mature polypeptide lacking the secretory peptide.

[0062] The term "propeptide" or "pro-peptide" refers to a portion of a protein that is cleaved during maturation or activation. Once cleaved, the propeptide generally has no independent biological function.

[0063] As used herein, the terms "domain" and "region" may be used interchangeably with the term "module."

[0064] The term "catalytic domain" or "catalytic module" or "core" refers to a domain of an enzyme that may or may not have been modified or altered, but that retains at least a portion of its original activity. The catalytic module region of a xylanase mutant polypeptide according to the present disclosure corresponds to amino acids that align with amino acids 1-191 of SEQ ID NO:1.

[0065] The term "linker" or "spacer" refers to a polypeptide comprising at least two amino acids that may be present between the domains of a multidomain protein, e.g., an enzyme comprising a catalytic domain and a binding domain such as a carbohydrate binding module (CBM), or any other enzyme hybrid, or between two proteins or polypeptides produced as a fusion polypeptide, e.g., a fusion protein comprising two core enzymes. For example, a fusion protein of a catalytic domain and a CBM is provided by sequentially fusing the DNA sequence encoding the catalytic domain, the DNA sequence encoding the linker, and the DNA sequence encoding the CBM into one open reading frame and expressing this construct.

[0066] The following abbreviations are used for amino acids: A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valin W Trp Tryptophan Y Tyr Tyrosine

[0067] Substitutions are described using the following nomenclature: amino acid residue in the protein backbone; position; substituted amino acid residue. According to this nomenclature, for example, a single residue substitution at position 23 from an alanine to a tyrosine residue is designated Ala23Tyr or A23Y.

[0068] As used herein, the term "comprising" includes the broader meanings of "including," "containing," and "comprehending," as well as the narrower terms "consisting of" and "consisting only of."

[0069] As used herein, "expression" includes any step involved in producing a polypeptide in a host cell, including, but not limited to, transcription, translation, post-translational modification and secretion. Expression may be followed by harvesting, i.e., recovery of the host cell or the expression product.

[0070] As used herein, the term "silage" is a type of fodder or animal feed made from preserved plant-based material.

[0071] In certain embodiments, the mutant polypeptide has xylanase activity and an amino acid sequence that has 79% or more, 81% or more, 82% or more, 84% or more, 87% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to amino acids 1-191 of SEQ ID NO:1. In certain embodiments, the mutant polypeptide does not have 100% sequence identity to amino acids 1-191 of SEQ ID NO:1. In certain embodiments, the amino acid numbering of the mutant polypeptide corresponds to the amino acid numbering of SEQ ID NO:1. In alternative embodiments, the amino acid numbering of the mutant polypeptide corresponds partially to the amino acid numbering of SEQ ID NO:1.

[0072] In one embodiment, the amino acid sequence of the mutant polypeptide has an amino acid sequence identity of 84% or more, preferably 85% or more, more preferably 86% or more, more preferably 87% or more, more preferably 88% or more, even more preferably 89% or more, and most preferably 90% or more, but less than 100%, to amino acids 1 to 191 of SEQ ID NO:1.

[0073] In one embodiment, a variant polypeptide is disclosed that comprises an amino acid sequence having 84% or more but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1, which amino acid sequence has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0074] In one embodiment, a variant polypeptide is disclosed that comprises an amino acid sequence having 85% or more but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1, which has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0075] In one embodiment, a variant polypeptide is disclosed that comprises an amino acid sequence having 86% or more but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1, which has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0076] In one embodiment, a variant polypeptide is disclosed that comprises an amino acid sequence having 87% or more but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1, which has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0077] In one embodiment, a variant polypeptide is disclosed that comprises an amino acid sequence having 88% or more but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1, which has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0078] In one embodiment, a variant polypeptide is disclosed that comprises an amino acid sequence having 89% or more but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1, which amino acid sequence has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0079] In one embodiment, a variant polypeptide is disclosed that comprises an amino acid sequence having 90% or more but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1, which has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0080] In one embodiment, a variant polypeptide is disclosed that comprises an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1, which amino acid sequence has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0081] In one embodiment, the mutant polypeptide comprises an amino acid sequence that has at least 79% amino acid sequence identity with amino acids 1 to 191 of SEQ ID NO:1, preferably at least 82%, more preferably at least 84%, even more preferably at least 87%, and most preferably at least 90% but less than 100%, and the amino acids of the mutant polypeptide corresponding to amino acids 31 to 191 of SEQ ID NO:1 have at least 90% sequence identity with amino acids 31 to 191 of SEQ ID NO:1, preferably at least 95%, more preferably at least 99%, and even more preferably at least 100% sequence identity.

[0082] In certain embodiments, the mutant polypeptide has xylanase activity; amino acids 1-191 of SEQ ID NO:1 with amino acid substitutions at positions 23 or 28, or positions 23 and 28, or positions corresponding to positions 23 and 28 of SEQ ID NO:1; and It has at least one additional amino acid substitution that results in a disulfide bridge between two Cys residues within the 1-191 amino acid region.

[0083] In certain embodiments, the variant polypeptide has xylanase activity and an amino acid sequence that has at least 79%, at least 81%, at least 82%, at least 84%, at least 87%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids D1 to L220 of SEQ ID NO: 1 of the parent AM24 xylanase polypeptide. In certain embodiments, the variant polypeptide has xylanase activity and an amino acid sequence that has less than 79% sequence identity to amino acids D1 to L220 of SEQ ID NO: 1 of the parent AM24 xylanase polypeptide.

[0084] In one embodiment, the mutant polypeptide comprises residues 3C and 30C, which are positions corresponding to positions 3 and 30 of SEQ ID NO:1, and a disulfide bridge of the mutant polypeptide is formed between residues 3C and 30C.

[0085] In one embodiment, the mutant polypeptide comprises the amino acid substitutions T3C or T30C, which are at positions corresponding to positions 3 and 30 of SEQ ID NO:1, and at least one disulfide bridge of the mutant polypeptide is formed between the substituted residues.

[0086] In one embodiment, the variant polypeptide has a combination of amino acid substitutions: T3C and T30C, or T3C and N30C, or T3C and E30C, or T3C and Y30C, or T3C and D30C, or V3C and T30C, or V3C and N30C, or V3C and E30C, or V3C and Y30C, or V3C and D30C, or A3C and T30C, or A3C and N30C, or A3C and E30C, or A3C and Y30C, or A3C and D30C, or P3C and T30C, or P3C and N30C, or P3C and E30C, or P3C and Y30C, or P3C and D30C, wherein the substituted positions correspond to positions 3 and 30 of SEQ ID NO:1, and at least one disulfide bridge of the mutant polypeptide is formed between the two substituted residues.

[0087] In another embodiment, the mutant polypeptide comprises disulfide bridges between Cys residues within the 1-191 amino acid region of the mutant polypeptide, and the positions of those Cys residues are different from the positions corresponding to positions 3 and 30 of SEQ ID NO:1.

[0088] In one embodiment, the mutant polypeptide comprises at least one disulfide bridge between Cys residues within a 1-30 amino acid region of the mutant polypeptide, the positions of which correspond to positions 1 and 30 of SEQ ID NO:1.

[0089] In one embodiment, the cysteine ​​residues in the amino acid region of the variant polypeptide having an amino acid sequence identity of 79% or more but less than 100% with amino acids 1-191 of SEQ ID NO: 1 are obtained, for example, by variant gene synthesis, whereby the resulting disulfide bridge between said cysteine ​​residues is not naturally occurring. In one embodiment, the cysteine ​​residues are obtained by genetic modification of the parent gene, for example, by site-directed mutagenesis, whereby the resulting disulfide bridge between said cysteine ​​residues is also not naturally occurring. Using a number of protein engineering approaches or even gene synthesis, it may be possible to create amino acid substitutions at specific positions in the amino acid chain, replacing a natural or wild-type amino acid at a specific position with another amino acid residue. In one embodiment, at least two amino acid residues in the 1-191 amino acid region of the variant polypeptide are substituted with cysteines, thereby allowing the formation of disulfide bonds between the sulfur atoms of the substitute cysteine ​​residues. In another embodiment, the variant polypeptide comprises cysteine ​​residues in at least two alternative positions that allow the formation of disulfide bridges within the 1-191 amino acid region of the variant polypeptide. In yet another embodiment, the variant polypeptide comprises cysteine ​​residues that allow for the formation of more than one disulfide bridge in the 1-191 amino acid region of the variant polypeptide.

[0090] In one embodiment, at least one disulfide bridge within the 1-191 amino acid region stabilizes the mutant polypeptide, thereby increasing the pH stability and thermal stability of the mutant polypeptide compared to mutant AM24 lacking a disulfide bridge.

[0091] In certain embodiments, the variant polypeptide comprises one or more amino acid substitutions at positions 23 or 28, or at positions 23 and 28.

[0092] In one embodiment, the mutant polypeptide comprises at least one disulfide bridge between Cys residues within a 1-30 amino acid region of the mutant polypeptide, and at least one amino acid substitution at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 1-30 of SEQ ID NO:1.

[0093] In certain embodiments, the mutant polypeptide comprises at least one amino acid substitution at A23, S23 or S28, or at A23 or S23 and S28.

[0094] In one embodiment, the mutant polypeptide comprises at least one disulfide bridge between two Cys residues within the 1-191 region, as well as at least one amino acid substitution at positions A23 or S28, or at positions A23 and S28, which correspond to positions 23 and 28 of SEQ ID NO:1.

[0095] In an alternative embodiment, the variant polypeptide comprises at least one disulfide bridge between two Cys residues within the 1-191 region, as well as at least one amino acid substitution at S23 or S28, or at S23 and S28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0096] In certain embodiments, the substituted amino acid residues at positions 23 or 28, or positions 23 and 28, are different from the original amino acid residues at those positions. In certain embodiments, the amino acid substitutents are selected from the group consisting of H, I, L, M, N, P, Q, R, S, T, V, W, or Y, provided that the substitute amino acid residue is different from the naturally occurring amino acid residue at that position.

[0097] In one embodiment, the mutant polypeptide is Substitution of the amino acid at position 23 with H, M, P, S, T, R, W, or Y; or Substitution of the amino acid at position 28 with I, L, M, N, P, Q, R, T, V, W or Y; or any combination thereof.

[0098] In one embodiment, the mutant polypeptide is Substitution of the amino acid at position 23 with H, M, P, S, T, R, W, or Y; or Substitution of the amino acid at position 28 with I, L, M, N, Q, R, T, V, W or Y; or any combination thereof.

[0099] In certain embodiments, the mutant polypeptide comprises the amino acid substitution A23H, A23M, A23P, A23S, A23T, A23R, A23W, or A23Y.

[0100] In certain embodiments, the mutant polypeptide comprises the amino acid substitutions S23H, S23M, S23P, S23T, S23R, S23W, or S23Y.

[0101] In certain embodiments, the mutant polypeptide comprises the amino acid substitution S28I, S28L, S28M, S28N, S28P, S28Q, S28R, S28T, S28V, S28W, or S28Y.

[0102] In one embodiment, the variant polypeptide has a combination of amino acid substitutions: A23H and S28I, A23H and S28L, A23H and S28M, A23H and S28N, A23H and S28P, A23H and S28Q, A23H and S28R, A23H and S28T, A23H and S28V, A23H and S28W, A23H and S28Y, A23M and S28I, A23M and S28L, A23M and S28M, A23M and S28N, A23M and S28P, A23M and S28Q, A23M and S28R, A23M and S28T, A23M and S28V, A23M and S28W, A23M and S28Y, A23P and S28I, A23P and S28L, A23P and S28M, A23P and S28N, A23P and S28P, A23P and S28Q, A23P and S28R, A23P and S28T, A23P and S28V, A23P and S28W, A23P and S28Y, A23S and S28I, A23S and S28L, A23S and S28M, A23S and S28N, A23S and S28P, A23S and S28Q, A23S and S28R, A23S and S28T, A23S and S28V, A23S and S28W, A23S and S28Y, A23T and S28I, A23T and S28L, A23T and S28M, A23T and S28N, A23T and S28P, A23T and S28Q, A23T and S28R, A23T and S28T, A23T and S28V, A23T and S28W, A23T and S28Y, A23R and S28I, A23R and S28L, A23R and S28M, A23R and S28N, A23R and S28P, A23R and S28Q, A23R and S28R, A23R and S28T, A23R and S28V, A23R and S28W, A23R and S28Y, A23W and S28I, A23W and S28L, A23W and S28M, A23W and S28N, A23W and S28P, A23W and S28Q, A23W and S28R, A23W and S28T, A23W and S28V, A23W and S28W, A23W and S28Y, A23Y and S28I, A23Y and S28L, A23Y and S28M, A23Y and S28N, A23Y and S28P, A23Y and S28Q, A23Y and S28R, A23Y and S28T, A23Y and S28V, A23Y and S28W, A23Y and S28Y, S23H and S28I, S23H and S28L, S23H and S28M, S23H and S28N, S23H and S28P, S23H and S28Q, S23H and S28R, S23H and S28T, S23H and S28V, S23H and S28W, S23H and S28Y, S23M and S28I, S23M and S28L, S23M and S28M, S23M and S28N, S23M and S28P, S23M and S28Q, S23M and S28R, S23M and S28T, S23M and S28V, S23M and S28W, S23M and S28Y, S23P and S28I, S23P and S28L, S23P and S28M, S23P and S28N, S23P and S28P, S23P and S28Q, S23P and S28R, S23P and S28T, S23P and S28V, S23P and S28W, S23P and S28Y, S23T and S28I, S23T and S28L, S23T and S28M, S23T and S28N, S23T and S28P, S23T and S28Q, S23T and S28R, S23T and S28T, S23T and S28V, S23T and S28W, S23T and S28Y, S23R and S28I, S23R and S28L, S23R and S28M, S23R and S28N, S23R and S28P, S23R and S28Q, S23R and S28R, S23R and S28T, S23R and S28V, S23R and S28W, S23R and S28Y, S23W and S28I, S23W and S28L, S23W and S28M, S23W and S28N, S23W and S28P, S23W and S28Q, S23W and S28R, S23W and S28T, S23W and S28V, S23W and S28W, S23W and S28Y, Includes S23Y and S28I, S23Y and S28L, S23Y and S28M, S23Y and S28N, S23Y and S28P, S23Y and S28Q, S23Y and S28R, S23Y and S28T, S23Y and S28V, S23Y and S28W or S23Y and S28Y.

[0103] In preferred embodiments, the variant polypeptides have improved stability at elevated temperatures.

[0104] In preferred embodiments, the variant polypeptides have improved thermal and / or pH stability at elevated temperatures.

[0105] In a preferred embodiment, the mutant polypeptide comprises the amino acid substitutions A23S and S28I together with at least one disulfide bridge between two Cys residues within the 1-191 amino acid region and has improved stability and / or activity compared to the parent xylanase AM24.

[0106] In a preferred embodiment, the mutant polypeptide comprises the amino acid substitutions A23S and S28I together with at least one disulfide bridge between two Cys residues in the 1-191 amino acid region and has improved thermostability and / or pH stability compared to the parent xylanase AM24. In an embodiment, the mutant polypeptide comprises the amino acid substitutions A23S and S28I together with at least one disulfide bridge between two Cys residues in the 1-191 amino acid region and has higher xylanase activity at elevated temperatures of 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, compared to the parent xylanase AM24. In an embodiment, the mutant polypeptide comprises the amino acid substitutions A23S and S28I together with at least one disulfide bridge between two Cys residues in the 1-191 amino acid region and has improved xylanase activity retention at pH 7 or higher and at temperatures of 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, compared to the parent xylanase AM24.

[0107] In a preferred embodiment, the mutant polypeptide comprises the amino acid substitutions A23S and S28I together with at least one disulfide bridge between two Cys residues in the 1-30 amino acid region and has improved stability and / or activity compared to the parent xylanase AM24. In a preferred embodiment, the mutant polypeptide comprises the amino acid substitutions A23S and S28I together with at least one disulfide bridge between two Cys residues in the 1-30 amino acid region and has improved thermostability and / or pH stability compared to the parent xylanase AM24. In a preferred embodiment, the mutant polypeptide comprises the amino acid substitutions A23S and S28I together with at least one disulfide bridge between two Cys residues in the 1-30 amino acid region and has higher xylanase activity at elevated temperatures of 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, when compared to the parent xylanase AM24. In one embodiment, the mutant polypeptide comprises amino acid substitutions A23S and S28I together with at least one disulfide bridge between two Cys residues within the 1-30 amino acid region and has improved retention of xylanase activity at pH 7 or above and at temperatures 70°C or above, preferably 80°C or above, more preferably 90°C or above, when compared to the parent xylanase AM24.

[0108] In one embodiment, the mutant polypeptide comprises only one amino acid substitution A23S or S28I with at least one disulfide bridge between two Cys residues in the 1-191 amino acid region and has improved stability and / or activity compared to parent xylanase AM24. In one embodiment, the mutant polypeptide comprises only one amino acid substitution A23S or S28I with at least one disulfide bridge between two Cys residues in the 1-191 amino acid region and has improved thermostability and / or pH stability compared to parent xylanase AM24. In one embodiment, the mutant polypeptide comprises only one amino acid substitution A23S or S28I with at least one disulfide bridge between two Cys residues in the 1-191 amino acid region and has higher xylanase activity at elevated temperatures of 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, compared to parent xylanase AM24. In one embodiment, the mutant polypeptide comprises only one amino acid substitution A23S or S28I together with at least one disulfide bridge between two Cys residues within the 1-191 amino acid region and has improved retention of xylanase activity at pH 7 or above and temperatures of 70°C or above, preferably 80°C or above, more preferably 90°C or above, when compared to the parent xylanase AM24.

[0109] In certain embodiments, the mutant polypeptide comprises an amino acid substitution at position 23 and / or position 28, lacks a disulfide bridge within the 1-191 amino acid region, and has improved stability and / or activity compared to parent xylanase AM24. In certain embodiments, the mutant polypeptide comprises an amino acid substitution at position 23 and / or position 28, lacks a disulfide bridge within the 1-191 amino acid region, and has improved thermostability and / or pH stability compared to parent xylanase AM24. In certain embodiments, the mutant polypeptide comprises an amino acid substitution at position 23 and / or position 28, lacks a disulfide bridge within the 1-191 amino acid region, and has higher xylanase activity at elevated temperatures of 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, when compared to parent xylanase AM24. In one embodiment, the variant polypeptide comprises amino acid substitutions at positions 23 and / or 28, lacks disulfide bridges within the 1-191 amino acid region, and has improved retention of xylanase activity at pH 7 or above and at temperatures 70°C or above, preferably 80°C or above, and more preferably 90°C or above, when compared to parent xylanase AM24.

[0110] In certain embodiments, the variant polypeptide comprises at least one disulfide bridge between two Cys residues in the 1-191 amino acid region without further substitutions in the amino acid chain and has improved stability and / or activity compared to the parent xylanase AM24. Such variants may have lower stability and / or activity than variant polypeptides comprising additional substitutions at positions 23 or 28 or at positions 23 and 28. In certain embodiments, the variant comprises a disulfide bridge between the substituted residues T3C, T30C and has improved stability and / or activity compared to the parent xylanase AM24. In certain embodiments, the variant polypeptide comprises at least one disulfide bridge between two Cys residues in the 1-191 amino acid region without further substitutions in the amino acid chain and has improved thermal stability and / or pH stability compared to the parent xylanase AM24. In certain embodiments, the variant polypeptide comprises at least one disulfide bridge between two Cys residues in the 1-191 amino acid region without further substitutions in the amino acid chain and has higher xylanase activity at elevated temperatures of 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, when compared to the parent xylanase AM24. In certain embodiments, the variant polypeptide comprises at least one disulfide bridge between two Cys residues in the 1-191 amino acid region without further substitutions in the amino acid chain and has improved retention of xylanase activity at a pH of at least 7 and at a temperature of at least 70° C., preferably at least 80° C., more preferably at least 90° C., when compared to the parent xylanase AM24.

[0111] In certain embodiments, the mutant polypeptide has xylanase activity and comprises at least one additional amino acid substitution at a position other than 23 or 28, which in certain embodiments further improves the stability and / or activity of the mutant polypeptide. Thus, the claimed substitutions at positions 23 and / or 28 of the amino acid chain, together with the disulfide bridge between the two Cys residues in the 1-191 region, may be used as is or with additional substitution positions to improve the performance and stability of the parent xylanase AM24. In certain embodiments, the mutant polypeptide has xylanase activity and comprises at least one additional amino acid substitution at a position other than 23 or 28, which in certain embodiments further improves the thermostability and / or pH stability and / or xylanase activity retention of the mutant polypeptide. Thus, the claimed substitutions at positions 23 and / or 28 of the amino acid chain, together with the disulfide bridge between two Cys residues in the 1-191 region, can be used as is or together with additional substitution positions to improve the performance and thermal and / or pH stability of the parent xylanase AM24.

[0112] In certain embodiments, the total number of substitutions in the variant polypeptide compared to SEQ ID NO:1 is between 3 and 10, or between 4 and 10. In certain embodiments, the variant has 3 substitutions, or the variant has 4 substitutions, or the variant has 5 substitutions, or the variant has 6 substitutions, or the variant has 7 substitutions, or the variant has 8 substitutions, or the variant has 9 substitutions, or the variant has 10 substitutions.

[0113] In certain embodiments, the variant polypeptide or functional fragment has a predicted molecular weight of between 19 kDa and 26 kDa, preferably between 20 kDa and 25 kDa, which includes the amino acids that constitute the core region of the variant polypeptide, and does not include the signal sequence that is cleaved from the secreted variant polypeptide and the putative pro-sequence that is cleaved during maturation of the variant polypeptide. The predicted molecular weight may be determined by calculating the sum of the molecular weights of the individual amino acids within the variant polypeptide or functional fragment thereof.

[0114] In certain embodiments, the mutant polypeptide has improved thermal stability and / or pH stability as compared to a polypeptide having the sequence of SEQ ID NO: 1. In certain embodiments, the mutant polypeptide has improved thermal stability and pH stability as compared to a polypeptide having the sequence of SEQ ID NO: 1. In certain embodiments, the mutant polypeptide has improved thermal stability or improved pH stability as compared to a polypeptide having the sequence of SEQ ID NO: 1.

[0115] In certain embodiments, the mutant polypeptides have higher xylanase activity when compared to a polypeptide having the sequence of SEQ ID NO: 1. In certain embodiments, the mutant polypeptides have improved retention of xylanase activity when compared to a polypeptide having the sequence of SEQ ID NO: 1. In certain embodiments, the mutant polypeptides have improved pH-dependent retention of xylanase activity and temperature-dependent retention of xylanase activity when compared to a polypeptide having the sequence of SEQ ID NO: 1.

[0116] In certain embodiments, at least one disulfide bridge between two Cys residues in the 1-191 amino acid region and an amino acid substitution at positions 23 or 28 or 23 and 28 of the mutant polypeptide improves the thermal and / or pH stability of the mutant polypeptide when compared to a polypeptide having the sequence of SEQ ID NO: 1. In certain embodiments, at least one disulfide bridge between two Cys residues in the 1-191 amino acid region of the mutant polypeptide improves the thermal and / or pH stability of the mutant polypeptide when compared to a polypeptide having the sequence of SEQ ID NO: 1.

[0117] In one embodiment, an amino acid substitution at positions 23 or 28, or at positions 23 and 28, of a mutant polypeptide improves the thermal and / or pH stability of the mutant polypeptide when compared to a polypeptide having the sequence of SEQ ID NO:1.

[0118] In one embodiment, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region and amino acid substitutions at positions 23 or 28, or at positions 23 and 28 of the mutant polypeptide improve retention of xylanase activity of the mutant polypeptide when compared to a polypeptide having the sequence of SEQ ID NO:1.

[0119] In certain embodiments, the variant polypeptides have improved stability and / or activity compared to a polypeptide having a sequence of SEQ ID NO:1 in reaction conditions where the temperature is 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, and most preferably 96° C. or higher. In certain embodiments, the variant polypeptides have improved thermal stability and / or pH stability compared to a polypeptide having a sequence of SEQ ID NO:1 in reaction conditions where the temperature is 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, and most preferably 96° C. or higher. In certain embodiments, the variant polypeptides have improved retention of xylanase activity compared to a polypeptide having a sequence of SEQ ID NO:1 in reaction conditions where the temperature is 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, and most preferably 96° C. or higher. In certain embodiments, the variant polypeptides have improved stability and / or activity compared to a polypeptide having a sequence of SEQ ID NO:1 in reaction conditions where the pH is 2 or higher, 3 or higher, 3.5 or higher, 4 or higher, 4.5 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, or 11 or higher. In certain embodiments, the variant polypeptides have improved thermal and / or pH stability when compared to a polypeptide having a sequence of SEQ ID NO: 1 in reaction conditions where the pH is 2 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more. In certain embodiments, the variant polypeptides have improved retention of xylanase activity when compared to a polypeptide having a sequence of SEQ ID NO: 1 in reaction conditions where the pH is 2 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more. In certain embodiments, the variant polypeptides have improved thermal and / or pH stability at temperatures of 90° C. or more and / or at a pH of 7 or more when compared to the parent xylanase AM24.

[0120] The provision of xylanases that retain activity at temperatures above ambient temperature is advantageous for applications requiring xylan degradation at such reaction conditions. The high temperature resistance of xylanases has the advantage of high specific activity and enzyme stability. The use of high reaction temperatures also prevents, for example, contamination and increases mass transfer rates by reducing fluid viscosity when substrate concentrations are high. The use of highly stable xylanases reduces or avoids the need to modify commonly used process conditions.

[0121] Additionally, xylanases according to the present disclosure have improved stability and xylanase activity in acidic and / or neutral and / or alkaline conditions, which is advantageous, for example, in bleaching pulp in alkaline or acidic process conditions. In certain embodiments, xylanases according to the present disclosure have improved thermostability and / or pH stability and / or improved xylanase activity retention in acidic and / or neutral and / or alkaline conditions, which is advantageous, for example, in bleaching pulp in alkaline or acidic process conditions.

[0122] In certain embodiments, the degradation or modification of xylan is carried out in an aqueous environment in which xylanases are active.

[0123] In certain embodiments, the mutant xylanase polypeptide randomly hydrolyzes accessible β-1,4-D-xylosidic bonds.

[0124] In one embodiment, the parent xylanase of the mutant polypeptide of the first aspect is one of the AM24 xylanase homologs of Figures 1A and 1 B. In one embodiment, the parent of the mutant polypeptide of a xylanase having 79% or more but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO: 1 is any of the homologs of xylanase AM24 of Figures 1A and 1 B.

[0125] In one embodiment, the mutant polypeptide of the first aspect also includes a mutant of an AM24 xylanase homolog, which mutant of an AM24 xylanase homolog has an amino acid sequence identity of 79% or more but less than 100% to amino acids 1 to 191 of SEQ ID NO:1.

[0126] In one embodiment, the variant of AM24 xylanase homolog has an amino acid sequence identity of 79% or more but less than 100% with amino acids 1-191 of SEQ ID NO:1, and the amino acid sequence of the variant of AM24 xylanase homolog has xylanase activity, at least one disulfide bridge between two Cys residues within the 1-191 amino acid region, and amino acid substitutions at positions corresponding to positions 23 or 28, or positions 23 and 28, or positions 23 and 28 of SEQ ID NO:1.

[0127] In certain embodiments, variants of AM24 xylanase homologs have improved stability and / or xylanase activity compared to the parent AM24 xylanase homolog. In certain embodiments, variants of AM24 xylanase homologs have improved stability and / or xylanase activity compared to the polypeptide having the sequence of SEQ ID NO:1. In certain embodiments, variants of AM24 xylanase homologs have improved thermostability and / or pH stability and / or improved retention of xylanase activity compared to the parent AM24 xylanase homolog. In certain embodiments, variants of AM24 xylanase homologs have improved thermostability and / or pH stability and / or improved retention of xylanase activity compared to the polypeptide having the sequence of SEQ ID NO:1.

[0128] In certain embodiments, variants of AM24 xylanase homologs have improved stability and / or xylanase activity compared to a polypeptide having a sequence of SEQ ID NO:1 and / or compared to the parent AM24 xylanase homolog in reaction conditions where the temperature is 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, and most preferably 96° C. or higher. In certain embodiments, variants of AM24 xylanase homologs have improved stability and / or xylanase activity compared to a polypeptide having a sequence of SEQ ID NO:1 and / or compared to the parent AM24 xylanase homolog in reaction conditions where the pH is 2 or higher, 3 or higher, 3.5 or higher, 4 or higher, 4.5 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, or 11 or higher. In certain embodiments, variants of AM24 xylanase homologs have improved thermostability and / or pH stability and / or improved retention of xylanase activity when compared to a polypeptide having a sequence of SEQ ID NO:1 and / or when compared to the parent AM24 xylanase homolog in reaction conditions where the temperature is 70° C. or higher, preferably 80° C. or higher, more preferably 90° C. or higher, and most preferably at least 96° C. or higher. In certain embodiments, variants of AM24 xylanase homologs have improved thermostability and / or pH stability and / or improved retention of xylanase activity when compared to a polypeptide having a sequence of SEQ ID NO:1 and / or when compared to the parent AM24 xylanase homolog in reaction conditions where the pH is 2 or higher, 3 or higher, 3.5 or higher, 4 or higher, 4.5 or higher, 5 or higher, 6 or higher, 7 or higher, 8 or higher, 9 or higher, 10 or higher, or 11 or higher.

[0129] In some embodiments of the second aspect of the present disclosure, the present mutant polypeptide of xylanase is part of a recombinant polypeptide that is a fusion protein. The fusion protein may have other catalytic or binding activities in addition to the activity of xylanase. In some embodiments, the mutant polypeptide of xylanase is connected to additional peptides and / or polypeptides using a linker.

[0130] In some embodiments, the fusion protein comprises a signal sequence that is advantageous in the translocation of the recombinant polypeptide in and out of the host cell. In some embodiments, the signal sequence may or may not comprise a carrier polypeptide sequence. In some embodiments, the fusion protein comprises a carbohydrate binding moiety (CBM) as a carrier polypeptide, which is optionally a fragment of another protein or enzyme from the same or a different organism as the xylanase variant polypeptide. The signal sequence and the carrier polypeptide may be from the same host or from different hosts. In some embodiments, the fusion protein comprises a signal sequence from the same host cell and a fusion protein comprising a xylanase variant polypeptide is produced. In some embodiments, the signal sequence is from the same filamentous fungal host and a fusion protein comprising a xylanase variant polypeptide is produced. In another embodiment, the signal sequence is from another organism.

[0131] In certain embodiments, the fusion protein contains an amino acid sequence that facilitates purification, such as a His tag or a polyhistidine tag, which includes a stretch of histidine residues.

[0132] In certain embodiments, the fusion protein comprises an amino acid sequence having enzymatic activity, said sequence having enzymatic activity being not the same sequence as the amino acid sequence encoding the variant polypeptide having xylanase activity.

[0133] In certain embodiments, the fusion protein includes an amino acid sequence of a carbohydrate binding moiety that provides binding affinity to the fusion protein.

[0134] In one embodiment, the enzyme composition comprises: at least one stabilizer selected from polyols, propylene glycol, polyethylene glycol, hexylene glycol, glycerol, sugars, sugar alcohols, polysaccharides, lactic acid, peptides, surfactants, or combinations thereof; or at least one preservative or buffer selected from organic acids, citric acid, ascorbic acid, benzoic acid and their salts and derivatives, sodium benzoate, benzoates, hydroxybenzoates and derivatives, phosphates, sorbic acid, sodium sorbate, sorbates, salts, sodium or potassium chloride, 1,2-benzisothiazolin-3-one (BIT), or combinations thereof; b. optionally, at least one inhibitor selected from boric acid, a boric acid derivative, an aromatic boric acid ester, 4-formylphenylboronic acid, a phenylboronic acid derivative, an inhibitory peptide compound, or a combination thereof; c. at least one enzyme selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, nucleases, pectinases, pectinolytic enzymes, pectate lyases, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases, or combinations thereof, optionally with or without a mediator; and d. Optionally, at least one filler selected from maltodextrin, flour, sodium chloride, sulfate, sodium sulfate, or combinations thereof. Further includes:

[0135] Components a-d provide the enzyme composition with improved properties. The enzyme composition is compatible with components a-d, improving the applicability of the enzyme composition in various applications. Salts such as sodium chloride and sodium sulfate function as drying aids.

[0136] In certain embodiments, an enzyme composition comprising a variant xylanase polypeptide and an additional enzyme is advantageous in providing a synergistic effect. Such an additional enzyme is desirable when the enzyme composition comprising a variant xylanase polypeptide is used, for example, in pulp or silage processing and feed or foodstuff preparation. Particularly advantageous synergistic enzymes that work with xylanases in pulp or silage processing are cellulases, hemicellulases, phytases and amylases. Particularly advantageous synergistic enzymes that work with xylanases in feed or foodstuff preparation are cellulases, hemicellulases, phytases and amylases.

[0137] In certain embodiments, the enzyme composition is provided in the form of a liquid or solid composition, such as a solution, dispersion, paste, powder, granules, granules, coated granules, tablets, cakes, crystals, crystal slurries, gels or pellets.

[0138] In certain embodiments, the enzyme preparation or composition further comprises other suitable additives selected from the group consisting of bleaches, anti-corrosive agents, builders, anti-resoiling agents, optical brighteners, dyes, pigments, fragrances, caustics, abrasives, and preservatives.

[0139] The present disclosure further relates to uses and methods of use of the enzyme compositions as disclosed herein to degrade xylan.

[0140] In one embodiment, a recombinant host cell is provided, the host cell comprising: a. a filamentous fungal cell of the phylum Ascomycota, subphylum Pezizomycotina; preferably a filamentous fungal cell of the group consisting of a member of the class Sordariomycetes or Eurotiomycetes, the subclass Hypocreomycetidae or Sordariomycetidae or Eurotiomycetidae, the order Hypocreales or Sordariales or Eurotiales, the family Hypocreacea or Nectriacea or Chaetomiaceae or Aspergillaceae, the genus Trichoderma (asexual form of Hypocrea), the genus Fusarium or Acremonium or the genus Humicola or the genus Thermothelomyces or the genus Myceliophthora or the genus Aspergillus; More preferably, Trichoderma reesei (Hypocrea jecorina), T.citrinoviridae, T.longibrachiatum, T.virens, T.harzianum, T.asperellum, T.atroviridae, T.parareesei, Fusarium filamentous fungal cells of the group consisting of the species oxysporum, F.gramineanum, F.pseudograminearum, F.venenatum, Acremonium(Cephalosporium)chrysogenum, Humicola insolens, Humicola grisea, Thermothelomyces thermophilus, Myceliophthora thermophila, Aspergillus niger, Aspergillus niger var. awamori and Aspergillus oryzae; b. bacterial cells, preferably gram-positive bacilli, such as B. subtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens, B. pumilus, gram-negative bacteria, such as Escherichia coli, actinomycetes, such as Streptomyces sp.; and cc yeasts, such as Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica selected from the group consisting of; Preferably, the host cell is a filamentous fungal cell such as Trichoderma or a Gram-positive bacillus such as Bacillus; most preferably, selected from Trichoderma reesei or Bacillus subtilis or B. pumilus or B. licheniformis.

[0141] In certain embodiments, the recombinant host cell is selected from the group consisting of a filamentous fungal cell (e.g., Trichoderma or Trichoderma reesei, Aspergillus or Aspergillus oryzae or Aspergillus niger, Thermothelomyces or Thermothelomyces heterothallica or Humicola or Humicola insolens or Fusarium or Fusarium venenatum), a bacterial cell, preferably a Gram-positive bacillus (e.g., Bacillus subtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens, B. pumilus), a Gram-negative bacterium (e.g., Escherichia coli), an Actinomycete (e.g., Streptomyces sp., Nonomuraea flexuosa) or a yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica).

[0142] Recombinant host cells can be used to produce and contain the polynucleotides encoding xylanase mutant polypeptides. Recombinant host cells can be operably linked to one or more regulatory sequences that direct the production of xylanase mutant polypeptides. Recombinant host cells are also useful in preparing xylanase mutants with different properties. For example, host cells can be selected that provide post-translational modifications that are beneficial for stability or activity, or that facilitate post-processing of the xylanase mutant polypeptides produced in the host cells.

[0143] In certain embodiments, the host cells are non-pathogenic, which is particularly advantageous when the host cells or their products are used in animal feed.

[0144] In an embodiment of the sixth aspect, the xylan-containing material is a plant-based or partially plant-based material. In another embodiment, the xylan-containing material is recycled waste paper; mechanical, chemical, semi-chemical, kraft or other paper pulp, wood or non-wood pulp, or fiber subjected to a fermentation refining process; or a material containing guar gum or locust bean gum.

[0145] In certain embodiments, the present variant xylanase polypeptides or the present enzyme compositions are used in pulp processing, such as pulp bleaching, to enhance cellulose hydrolysis and increase the efficiency of pulp purification. The present variant xylanase polypeptides are useful in enhancing lignin release from paper pulp and in reducing the use of harmful bleaching agents such as chlorine chemicals and sodium hydrosulfite.

[0146] In certain embodiments, the xylanase variant polypeptides or fusion proteins or enzyme compositions of the present disclosure are used in biomass processing and biomass hydrolysis, and wastewater remediation. The use of the present xylanase variant polypeptides is useful in the production of biological fuels, where xylanases can be used to convert lignocellulosic feedstocks from lignocellulosic biomass to biofuels such as ethanol.

[0147] In embodiments in which the present variant polypeptides or enzymes of xylanase are used in animal feed, the animal is a monogastric or ruminant animal, hi another embodiment, the animal is an aquaculture organism such as a broiler chicken, layer chicken, pig, piglet, turkey, or fish.

[0148] In some embodiments, the feed is an animal feed and comprises or consists of grains and cereals, such as corn and soybean meal, or wheat, oat, barley and / or rice. In embodiments in which the present variant polypeptides or enzymes of xylanase are used in animal feed, at least one xylan-containing product or by-product is provided in the feed. The feed in which the present variant polypeptides or enzymes of xylanase are used has improved nutritional value compared to a feed that does not contain a variant polypeptide of xylanase. The enzyme composition and the present variant polypeptides of xylanase degrade xylan present in the feed, thereby making the feed more digestible for animals. In particular, the enzyme composition and the present variant polypeptides of xylanase can digest xylan present in the feed, for example in a corn soybean-containing feed, which has a beneficial effect on gut microbes and thus on the performance of the animal. The animal feed in which the present variant polypeptides or enzymes of xylanase are used can be formulated in the form of a wet composition or a dry composition.

[0149] In embodiments in which the variant polypeptides or fusion proteins or enzyme compositions of the present disclosure are used in food industry processes or commercial applications, the processes or applications include, for example, the extraction of coffee, vegetable oils, xylose (for conversion to xylitol) and starch, the removal of gums from plant fiber sources, the baking and dough conditioner industries (e.g., bread production), and the beverage industry (alcoholic fermentation).

[0150] The use of the mutant polypeptide, the fusion protein and the enzyme composition is advantageous, for example, in baking, where it converts water-insoluble xylan into a soluble form, increasing water binding, reducing dough hardness and increasing volume.The fusion protein and the enzyme composition are also advantageous, for example, in the processing and manufacture of soy milk products, where it improves the yield, color, protein content and taste of soy milk products.The use of the mutant polypeptide, the fusion protein and the enzyme composition is advantageous, for example, in the beverage industry, where it improves the clarification process of beverages such as fruit juice and wine. EXAMPLES

[0151] Example 1. Design of AM24 (truncated Nf_Xyn11A) mutants Aiming to further improve stability, mutants were engineered from the thermophilic AM24 xylanase (SEQ ID NO: 1). AM24 is the name used for a truncated form of Nf_Xyn11A, Nonomuraea flexuosa Xyn11A. AM24 contains amino acids D44 to L263 of full-length wild-type Nf_Xyn11A, thus missing most of the carbohydrate-binding domain (CBM) and linker region of full-length wild-type Nf_Xyn11A (Leskinen et al., 2005; Paloheimo et al., 2007).

[0152] In these mutants, a disulfide (SS) bridge was constructed to the N-terminus by changing the natural amino acids at positions 3 and 30 of AM24 to cysteine ​​(C). Two additional positions in the N-terminal portion of the molecule were also designed to contain mutations at amino acid positions 23 and / or 28.

[0153] [Table 1]

[0154] Example 2. Design of mutants from close Nf Xyn11A homologues A blastp (protein-protein BLAST) search for sequences with closest homology to the Nf Xyn11A (AM24) core region was performed from the NCBI protein database of non-redundant protein sequences (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins). The amino acid sequence of Nf_Xyn11A core (amino acids 1-191 from SEQ ID NO: 1, representing the mature core region according to Leskinen et al., 2005) was used as the query sequence. The algorithm parameters were as follows: maximum target sequence: 100; expectation threshold: 10; word size: 6; matrix BLOSUM62; gap cost: yes: 11, extension: 1; composition adjustment: conditional composition score matrix adjustment. The sequences of the 100 homologs were downloaded and aligned using Clustal Omega multiple sequence alignment (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) with default settings. The lowest sequence identity to the AM24 core sequence was 79.0%.

[0155] An identity matrix (phylogenetic tree) was created from the AM24 core and the 100 closest homologs of AM24. The tree was generated from the aligned sequences using the Phylogenetic Tree program from the ClustalOmega website (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) and is shown in Figure 1A and Figure 1B. The database code for each xylanase sequence is included. Default settings were used for the alignment.

[0156] From the 100 AM24 xylanase homologs, six xylanases produced in Trichoderma reesei were selected. Furthermore, two mutants were designed from each of those six AM24 homologs. The xylanases selected for production are shown in Table 2. Based on the alignment of the above six amino acid sequences with the AM24 sequence (SEQ ID NO: 1), the amino acid sequences of the six AM24 homologs were trimmed from the N-terminus and / or C-terminus to represent the putative core region of each xylanase. The alignment of the AM24 sequence of the six AM24 xylanase homologs selected to be produced with the trimmed (core) sequences is shown in FIG. 2. The AM24 core sequence includes amino acids 1 to 191 of SEQ ID NO: 1.

[0157] From the core sequences of each of the six xylanases mentioned above, two types of AM24 xylanase homolog mutants were designed. In the first type of mutant, SS bridges were added to the N-terminus of the xylanase by changing the natural amino acids at positions 3 and 30 to cysteines. The second type of mutant was designed using the same strategy used to design the AM24 mutant C31-4 (Example 1, Table 1). These AM24 homolog mutants contain SS bridges (C at positions 3 and 30), and further change the amino acid at position 23 to serine and the amino acid at position 28 to isoleucine when these amino acids are not originally natural amino acids. Details of the mutants designed and the mutations made are listed in Table 2.

[0158] [Table 2]

[0159] The percentage identity between the above amino acid sequences (SEQ ID NO: 30, 33, 36, 39, 42 and 45) and the AM24 core region (amino acids 1-191 of SEQ ID NO: 1) ranged from 79.0 to 96.3%. The identity matrix was generated using the EMBOSS Needle Pairwise Sequence Alignment program (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ) with default settings (matrix BLOSUM62, gap open 10, gap extension 0.5, end gap penalty false, end gap open 10, end gap extension 0.5). The percent identity matrix is ​​shown in Table 3.

[0160] [Table 3]

[0161] Example 3. Construction of expression cassettes and plasmids for producing xylanases and xylanase variants AM24 was expressed from two constructs. The nucleotide sequence of SEQ ID NO: 48 corresponds to that contained in the expression cassette pALK1502 (Paloheimo et al., 2007). In SEQ ID NO: 56, the internal NruI site of the gene encoding AM24 has been silenced and there are few other modifications in those codons (see sequence listing for further details). However, SEQ ID NO: 48 and SEQ ID NO: 56 encode the same amino acid sequence (SEQ ID NO: 1). AM24 variants were synthesized using PCR and restriction / ligation methods using pALK1502 as a template or ordered as synthetic genes. All genes encoding AM24 homologs and their variants were ordered as synthetic genes. The codons used in the amino acid substitutions (i.e., mutations) of the ordered synthetic AM24 variants were selected based on the codon usage of Trichoderma reesei. The nucleotide sequences of the genes encoding AM24 homologs were codon-optimized according to the codon usage of Trichoderma reesei. To improve the yield of bacterial xylanases, sequences encoding mature xylanases were fused to sequences encoding carrier polypeptides in expression constructs. The carrier used was the T. reesei cellobiohydrolase II (CBHII) CBD (A) and hinge (B) regions from native CBHII as described for pALK1502 in Paloheimo et al. (2007). A sequence encoding a Kex2 protease cleavage site (RDKR) was inserted between the carrier and the xylanase-encoding sequence. SEQ ID NOs for the xylanase nucleotide sequences included in the expression cassettes are listed in Tables 1 and 2. The gene constructs were expressed using the T. reesei cbh1 (cellobiohydrolase 1) promoter. Transcription was terminated using either the T. reesei cbh1 or cbh2 terminators. Either the native or synthetic gene encoding Aspergillus nidulans acetamidase was included in the expression constructs as a transformation marker.

[0162] Example 4. Production of xylanases and xylanase mutants in Trichoderma reesei The xylanase expression constructs described above, either as expression cassettes or expression plasmids isolated from expression plasmids, were transformed into T. reesei protoplasts as described by Penttila et al. (1987) and Karhunen et al. (1993). Three to 20 transformants from each transformation were picked and sporulated on potato dextrose (PD) slants. Spores from the PD slants were used to inoculate shake flask cultures (50 mL volume). Cultivation was carried out for 7 days (30°C, 250 rpm) in cellulase induction medium. After the cultivation was terminated, samples were taken, the fungal mycelium was removed by centrifugation (4000 rpm, 10 min), and the culture supernatant was collected for analysis.

[0163] Xylanase production was analyzed using SDS-PAGE analysis and activity assay. Xylanase activity was measured according to Bailey et al. (1992) but using reaction conditions of pH 7 (instead of 5.3) and 70°C (instead of 50°C).

[0164] All samples showed protein bands of the expected molecular weight for the corresponding xylanases and xylanase variants in SDS-PAGE gels, and all xylanases also showed glycosylated forms in those gels.

[0165] Using the analytical method used, xylanase activity could be measured from the culture supernatants of all xylanases and xylanase mutants produced, except for XB6 and its mutants, for which no xylanase activity was obtained or the activity was below the detection level.

[0166] Example 5. Analysis of xylanases and xylanase mutants Three different analytical methods were used to investigate the stability of xylanases and xylanase mutants in culture supernatants: 1) The unfolding temperatures of all xylanases were analyzed using Prometheus NT.48 (NanoTemper Technologies GmbH). Analysis of AM24 and AM24 mutants was performed from samples containing purified xylanases in 0.2 M glycine-NaOH buffer adjusted to pH 10. The pH of the buffer containing the samples was 9.92 (pH of the samples ranged from 4.32 to 4.38). The unfolding temperatures of AM24 homologs and their mutants were analyzed directly from the culture supernatant. The temperature range used for the analysis was 20 to 110 °C (heating rate +1 °C / min). In addition, the unfolding temperatures of AM24 and AM24 mutants were also analyzed at low pH (3.5 and 4.5) in 200 mM Na-acetate buffer. 2) Thermostability was analyzed from selected samples by using a modified activity assay of Bailey et al. (1992) as follows: reaction times used were 5 and 60 min, reaction pH was 7, and reaction temperatures were 70, 80, 90, and 96° C. Additionally, thermostability was analyzed from AM24 and AM24 mutant samples at pH 8 and 9 using reaction times of 5 min at 70, 80, 90, and 96° C. and 60 min at 70 and 80° C. 3) After incubating AM24 and AM24 mutant samples at pH 11, 90°C for 60 min, residual xylanase activity was measured from those samples at pH 7, 70°C using a 5 min reaction time.

[0167] The unfolding temperature of AM24 xylanase at high pH (pH of the buffer was adjusted to 10) was 84.8°C (Table 4). All AM24 mutants, except NFX23, had higher unfolding temperatures than AM24. The unfolding temperatures of the AM24 mutants at high pH ranged from 87.6 to 104.3°C (67.5°C for NFX23).

[0168] [Table 4]

[0169] At pH 3.5, the unfolding temperature of AM24 was 61.4° C. and at pH 4.5 it was 75.2° C. The unfolding temperatures of the AM24 mutant C31-4 were obviously better than those of AM24 at low pH, being 84.1° C. at pH 3.5 and 105.2° C. at pH 4.5. This result indicates that the described mutations improve the stability of the parent xylanase in a wide pH range.

[0170] The unfolding temperatures of the AM24 homologs at high pH (buffer pH adjusted to 10) ranged from 63.6 to 78.1°C (Table 5). The unfolding temperatures of the AM24 homolog mutants were improved when an S-S bridge was added. However, even higher improvements in unfolding temperature were obtained when both an S-S bridge and additional mutations at positions 23 and / or 28 were included. The highest increase in unfolding temperature was +26.8°C for XB3M2 and +33.5°C for XB5M2 compared to the corresponding parent xylanases (Table 5).

[0171] [Table 5]

[0172] Thermostability was analyzed from culture supernatants of a selected set of AM24 and its mutants: mutants with only SS-bridge forming mutations (T3C and T30C; AM24-CC), mutants without SS-bridge forming mutations but with mutations at positions 23 and / or 28 (AM24-AS, AM24-SI, AM24-AS+SI), and mutants with mutations at positions 23 and / or 28 combined with SS-bridge forming cysteine ​​mutations (AM24-CC+AS, AM24-CC+SI, C31-4). The results of the analysis performed at pH 7 with reaction times of 5 and 60 min are shown in Figure 3A and Figure 3B, respectively. At the highest temperatures (90 and / or 96 °C), clear differences were detected between the thermostability of AM24 and its mutants, with the AM24 mutants being more thermostable than AM24. Mutations at positions 23 or 28 improved the thermostability of AM24. Combining these two mutations resulted in further improvements in thermostability. Inclusion of an S-S bridge at the N-terminus resulted in a clear improvement in the thermal stability of AM24. However, when mutations at positions 23 or 28 were combined with an S-S bridge in one molecule, further improvements in thermal stability were detected. The best thermal stability was achieved when both mutations at positions 23 and 28 were combined with an S-S bridge.

[0173] The thermal stability of AM24 and mutant C31-4 was further analyzed at higher pH values ​​of pH 8 and 9 using reaction times of 5 and 60 min. The reaction temperatures were 70, 80, 90 and 96°C for the 5 min assay (Figure 4A and Figure 4C) and 70 and 80°C for the 60 min assay (Figure 4B and Figure 4D). In the analysis performed at pH 8, the thermal stability of C31-4 in the 5 min assay was clearly better than that of AM24 at the highest tested temperatures of 90 and 96°C (Figure 4A) and in the 60 min assay at 80°C (Figure 4B). In the analysis at pH 9, the thermal stability of C31-4 in the 5 min assay was clearly better at all tested temperatures (Figure 4C) and in the 60 min assay at 80°C (Figure 4D).

[0174] In the third thermostability test performed, the residual xylanase activity was measured from AM24 and C31-4 culture supernatants after incubating the samples at pH 11 and 90 °C for 60 min. The analysis was performed using two buffer systems: Britton-Robinson and Na phosphate. The residual xylanase activity was measured using pH 7, 70 °C, and a reaction time of 5 min. C31-4 withstood the harsh conditions better than AM24. In Britton-Robinson buffer, approximately 20% of the original activity (samples BR / 1 and BR / 2) and in Na phosphate buffer, approximately 26% (samples NaP / 1 and NaP / 2) remained in the C31-4 samples, whereas only 2-3% of the original activity remained in the AM24 samples (Figure 5).

[0175] Example 6. Production of AM24 and C31-4 xylanases for applied research Trichoderma strains producing AM24 or C31-4 were cultivated in 30L bioreactors to produce materials for application testing. Fermentation was carried out as fed-batch culture in cellulase-inducing medium. After fermentation was completed, the mycelium was removed by a filtration process and the culture supernatant was collected and concentrated. The resulting enzyme preparation was used in bleaching tests.

[0176] The produced enzyme preparations of AM24 and C31-4 contained equal amounts of the corresponding xylanase enzymes, and more than half of the total protein in the enzyme preparations consisted of each xylanase. Equal amounts of AM24 and C31-4 xylanases were used in the bleaching tests.

[0177] Example 7. Bleaching of eucalyptus kraft pulp using xylanase pretreatment at high pH and temperature A short bleaching sequence, XD0E (X, enzyme; D0 (D zero), chlorine dioxide (ClO2); E, extraction with sodium hydroxide) was used to compare the bleaching efficiency of pretreatment with AM24 and C31-4 xylanases at high temperature (90°C). Pretreatment was performed at two pH values, pH ~8 and pH ~10. The pulp used was oxygen delignified eucalyptus kraft pulp from an Asian mill with a Kappa number of 15.5 and brightness of 38.0 ISO%. A constant ClO2 dosage (0.2 x Kappa number) was used. Bleaching tests were performed in bleaching reactors. An anchored Kemu reactor (batch size 0.15-1.0 kg, Tmax 90°C, consistency 25%) was used in the X and E stages, and an air-heated Hellsten reactor (batch size 0.3-0.8 kg, Tmax 85°C, consistency 12%) was used in the D0 stage.

[0178] Approximately 2 g total protein per ton of pulp was used. Both xylanase products contained similar relative amounts of xylanase protein (Example 6). Enzyme treatment time was 60 min.

[0179] After the enzyme stage, the enzyme was inactivated by adding hot water and immediately adjusting the pH of the pulp suspension to a pH value between 2.5 and 3. Brightness and Kappa number were analyzed after the E stage.

[0180] [Table 6]

[0181] Pretreatment with both xylanases AM24 and C31-4 improved the brightness of eucalyptus pulp. At pH 8, the pulp pretreated with C31-4 had a brightness 2.8 units higher than the reference pulp and 1.0 unit higher than the pulp pretreated with AM24. The active chlorine (aCl) usage per ΔKappa at pH 8 for the C31-4 treated pulp was 0.2 kg / t lower than the AM24 treated pulp, and the active chlorine usage per ΔBr was 0.07 kg / t lower, and 0.28 kg / t and 0.19 kg / t lower, respectively, than the reference pulp (Table 6).

[0182] The AM24 treatment did not provide any benefit compared to the reference pulp when xylanase pretreatment was performed at pH 11. However, the C31-4 treated pulp had 2.7 units higher brightness, 0.09 kg / t lower active chlorine (aCl) usage per ΔKappa, and 0.18 kg / t lower active chlorine usage per ΔBr than the reference pulp (Table 6).

[0183] The increase in brightness (%) detected was significant, indicating that the AM24 xylanase variant C31-4 can be used for pretreatment or treatment of pulp and other materials at high pH and temperature.

[0184] Example 8. Bleaching of softwood (SW) kraft pulp with C31-4 at high pH and temperature The efficiency of C31-4 in bleaching softwood pulp at high temperature and high pH was tested by using a short bleaching sequence XD0E (X, enzyme; D0 (D zero); ClO2, chlorine dioxide; E, extraction with sodium hydroxide) at constant ClO2 dosage (0.2 x kappa number). Bleaching tests were carried out in the full sequence in bleaching reactors. An anchored Kemu reactor (batch size 0.15-1.0 kg, Tmax 90°C, consistency 25%) was used in the X and E stages, and an air-heated Hellsten reactor (batch size 0.3-0.8 kg, Tmax 85°C, consistency 12%) was used in the D0 stage.

[0185] The pulp used in the trials was oxygen delignified SW Kraft from a Scandinavian mill with the following characteristics: Kappa number 25.6, viscosity 1140 ml / g and brightness 26.8 ISO%. The dosage of the C31-4 enzyme preparation was approximately 2 g total protein per tonne of pulp. The conditions used in the various bleaching stages are listed in Tables 7-9.

[0186] After stage X, the pulp was washed three times. The first wash was done with water at reaction temperature (90°C), followed by two washes with cold water (4°C). After the washing steps, a centrifugation process (2600 rpm, 10 s) was used to remove the water and pulp samples of about 10-15 g (measured oven dried) were collected for analysis. Additionally, dry matter analysis of the C31-4 treated samples was performed using an IR dryer to better control the pulp volume. The kappa number and brightness of the pulp samples were analyzed.

[0187] The results of the study are detailed in Table 10.

[0188] [Table 7]

[0189] [Table 8]

[0190] [Table 9]

[0191] [Table 10]

[0192] Softwood kraft pulp pretreated with C31-4 at pH 10.5 had 2.7 units higher corrected final brightness compared to the reference pulp (without enzyme) at the same 5.4% active chlorine consumption.

[0193] Example 9. Bleaching of softwood (SW) kraft pulp using C31-4 at high pH and high temperature compared to AM24 xylanase at neutral pH and low temperature The bleaching test using softwood pulp and conditions as in Example 8 was repeated with AM24 and C31-4 xylanases. The pulp was washed at pH 11 and 60°C for about 30 minutes before its characterization and experimentation. After the washing step, the pulp had a kappa number of 16.7, a viscosity of 1050 ml / g and a brightness of 32.6 ISO%. The dosage of both enzymes (C31-4 and AM24) was 2 g total protein per ton of pulp. The conditions used in the xylanase treatment, bleaching and extraction stages are listed in Tables 11-13. The two enzymes were applied at different pH and temperatures in the xylanase treatment stage (X stage) since AM24 xylanase is known to be inactive in such pulp mill conditions, whereas the C31-4 variant was expected to be able to function in these conditions (pH 10.5 / 90°C) based on previous experiments.

[0194] [Table 11]

[0195] [Table 12]

[0196] [Table 13]

[0197] The results of the experiment are included in Table 14.

[0198] [Table 14]

[0199] Scandinavian softwood kraft pulp pretreated with C31-4 at pH 10.5 / 90°C had 2.8 units higher brightness compared to the reference pulp (no enzyme) at identical conditions throughout the XD0E bleaching sequence. The results confirmed that C31-4 improves brightness at high pH and elevated temperature conditions. AM24 xylanase showed a brightness increase of 1.2 units in the X stage (pH 7 / 80°C) at the adjusted conditions and at the same conditions used for C31-4 in the D0 and E stages.

[0200] Example 10. Use of thermophilic xylanase variants in feed applications The described thermophilic xylanase variants can be used in feeding animals, such as pigs and broilers. They can be used alone or in combination with other enzymes, such as phytases and / or mannanases.

[0201] The effect of the recombinant xylanase variant of the present invention on the growth of broilers is examined. The ultrafiltrate of the fermentation broth containing the recombinant xylanase is dried and the target level is applied to pelleted broiler diet, either alone or in combination with other commercially available enzymes. A control diet without xylanase enzyme is fed. The diet is based on rye and wheat or corn as grains.

[0202] The initial body weight of the broilers is 38-42 g. The trial lasts for 5-6 weeks. Each treatment consists of a minimum of 6 replicates with 10 broilers each. In all cases, the diets are analyzed for moisture, crude protein, crude fiber, fat, ash and enzyme protein.

[0203] Five diets are prepared for testing with each selected xylanase variant, as described below, with the parent xylanase administered as the standard treatment. 1) No-replenishment control (BD) 2) BD+ selected xylanase parent enzyme 1-10mg / kg 3) BD + mutants of the parent xylanase above, at the same doses as above 3) BD + selected xylanase parent enzyme 1-10 mg / kg, different doses compared to 1). 4) BD + mutants of the parent xylanase above, at the same doses as above

[0204] Animal health and mortality are checked daily by visual inspection. Body weight gain (BW), feed intake (FI) and feed conversion ratio (FCR) are measured on days 0, 14, 21 and 35. FCR is calculated as total feed consumed divided by body weight gain during the same period. Evaluation of the efficacy of recombinant xylanase variants is based on comparison with animals fed the corresponding wild-type xylanase and unsupplemented control diets.

[0205] Due to the high unfolding temperature, C31-4 xylanase and other thermophilic xylanase variants designed using the same principles disclosed herein are suitable for all applications where high thermostability is required, such as wood pulp and non-wood pulp applications and feed applications where high temperatures are used in the pelleting stage. The xylanase variants disclosed herein are also suitable to tolerate and / or function in process conditions that are not optimal for the activity and function of the enzyme in question, such as alkaline or acidic pH.

[0206] The foregoing description has provided a complete and informative description of the best mode currently contemplated by the inventors for carrying out the present invention as non-limiting examples of specific implementations and embodiments. However, it will be apparent to those skilled in the art that the present invention is not limited to the details of the embodiments presented above, and may be carried out in other embodiments using equivalent means, or in combinations of various embodiments, without departing from the characteristics of the present invention.

[0207] Furthermore, some of the features of the exemplary embodiments disclosed above can be used advantageously without the corresponding use of other features. As a result, any suitable combination of embodiments and aspects can be formed. Any combination of aspects or embodiments as disclosed herein can also be performed without at least one non-essential feature disclosed in the aspect or embodiment.

[0208] The appended claims define the scope of protection. Any method, process, product, or apparatus disclosed in the specification or drawings but not covered by the claims should not be understood as an embodiment of the claimed invention but is provided as an example useful for understanding the claimed invention.

[0209] References TIFF2024501524000020.tif119162

Claims

1. A variant polypeptide comprising an amino acid sequence having 79% to 100% amino acid sequence identity with amino acids 1 to 191 of SEQ ID NO:1, The amino acid sequence is having at least one disulfide bridge between two Cys residues within the 1-191 amino acid region; and an amino acid substitution at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1; A mutant polypeptide having xylanase activity.

2. 2. The variant polypeptide of claim 1, wherein the amino acid sequence has 84% ​​to 100% amino acid sequence identity with amino acids 1 to 191 of SEQ ID NO:

1.

3. The mutant polypeptide of claim 1, wherein the mutant polypeptide comprises residues 3C and 30C, which are positions corresponding to positions 3 and 30 of SEQ ID NO:1, and the disulfide bridge of the mutant polypeptide is formed between residues 3C and 30C.

4. 4. The mutant polypeptide of any one of claims 1 to 3, comprising at least one amino acid substitution in A23, S23 or S28, or A23 or S23 and S28.

5. Substitution of the amino acid at position 23 with H, M, P, S, T, R, W or Y; or Substitution of the amino acid at position 28 with I, L, M, N, P, Q, R, T, V, W or Y; or Any combination of them The mutant polypeptide of any one of claims 1 to 3, comprising:

6. 4. The mutant polypeptide of any one of claims 1 to 3, wherein the mutant polypeptide has improved thermal and / or pH stability compared to the polypeptide having the sequence of SEQ ID NO:

1.

7. The amino acid sequence of the variant polypeptide of claim 1; an amino acid sequence providing a secretory signal sequence; an amino acid sequence that facilitates purification; an amino acid sequence that controls and enhances production of said recombinant polypeptide; an amino acid sequence encoding a protease cleavage site; an amino acid sequence having enzymatic activity; and / or an amino acid sequence that provides a fusion protein with binding affinity; At least one of A fusion protein comprising:

8. 10. An enzyme composition comprising the mutant polypeptide of claim 1 or the fusion protein of claim 7.

9. at least one stabilizer selected from a polyol, propylene glycol, polyethylene glycol, hexylene glycol, glycerol, a sugar, a sugar alcohol, a polysaccharide, lactic acid, a peptide, a surfactant, or a combination thereof; or at least one preservative or buffer selected from an organic acid, citric acid, ascorbic acid, benzoic acid and salts and derivatives thereof, sodium benzoate, benzoate, hydroxybenzoate and derivatives, phosphates, sorbic acid, sodium sorbate, sorbate, salts, sodium or potassium chloride, 1,2-benzisothiazolin-3-one (BIT), or a combination thereof; The above a. and the following b. at least one inhibitor selected from boric acid, boric acid derivatives, aromatic boric acid esters, 4-formylphenylboronic acid, phenylboronic acid derivatives, peptide compounds having inhibitory activity, or combinations thereof; c. at least one enzyme selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, nucleases, pectinases, pectinolytic enzymes, pectate lyases, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases, or combinations thereof, with or without a mediator; and d. at least one filler selected from maltodextrin, flour, sodium chloride, sulfate, sodium sulfate, or combinations thereof; The enzyme composition according to claim 8, further comprising at least one selected from the group consisting of:

10. The enzyme composition according to claim 8 in the form of a liquid or solid composition.

11. A recombinant host cell comprising genetic elements that enable it to produce at least one recombinant polypeptide comprising the mutant polypeptide of claim 1 or the fusion protein of claim 7.

12. The host cell comprises a. Filamentous fungal cells of the phylum Ascomycota, subphylum Pezizomycotina; b. a bacterial cell, and c. yeast, Selected from the group consisting of: The recombinant host cell of claim 11.

13. 10. A method for producing a mutant polypeptide of claim 1 or a fusion protein of claim 7, comprising the steps of: a. culturing the recombinant host cell of claim 11 under conditions that allow the production of the recombinant polypeptide; or a. and b. recovering the recombinant polypeptide.

14. 13. A method for degrading or modifying xylan-containing material, comprising treating the xylan-containing material with an effective amount of a mutant polypeptide of claim 1, or a fusion protein of claim 7.

15. 13. Use of the variant polypeptide of claim 1 or the fusion protein of claim 7 in industrial processes or commercial applications such as pulp or silage processing.

16. 10. Use of a mutant polypeptide according to claim 1 or a fusion protein according to claim 7 in the preparation of a feed or foodstuff.

17. A method for degrading or modifying xylan-containing material, comprising treating the xylan-containing material with an effective amount of the enzyme composition of claim 8.

18. Use of the enzyme composition of claim 8 in industrial processes or commercial applications such as pulp or silage processing.

19. Use of the enzyme composition of claim 8 in the preparation of a feed or foodstuff.

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