Xylanases, nucleic acids encoding them and methods for making and using them

Abstract

The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal β-1,4-xylosidic linkages or endo-β-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.

1 .- 133 . (canceled) 
     
     
         134 . An isolated, synthetic or recombinant polypeptide or peptide having a xylanase, a mannanase and/or a glucanase activity comprising:
 (a) an amino acid sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or has 100% (complete) sequence identity to SEQ ID NO:384, over a region of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, 150, 200, 250, 300 or more residues, or over the full length of the polypeptide or enzyme, and enzymatically active subsequences (fragments) thereof,   (b) the amino acid sequence of (a), and comprising at least one amino acid residue conservative substitution, and the polypeptide retains xylanase, a mannanase and/or a glucanase activity;   (c) the amino acid sequence of (b), wherein the conservative substitution comprises replacement of an aliphatic amino acid with another aliphatic amino acid; replacement of a serine with a threonine or vice versa; replacement of an acidic residue with another acidic residue; replacement of a residue bearing an amide group with another residue bearing an amide group; exchange of a basic residue with another basic residue; or, replacement of an aromatic residue with another aromatic residue, or a combination thereof,   and optionally the aliphatic residue comprises Alanine, Valine, Leucine, Isoleucine or a synthetic equivalent thereof; the acidic residue comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof; the residue comprising an amide group comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof; the basic residue comprises Lysine, Arginine or a synthetic equivalent thereof; or, the aromatic residue comprises Phenylalanine, Tyrosine or a synthetic equivalent thereof;   (d) the polypeptide of (a), (b), or (c) having a xylanase, a mannanase and/or a glucanase activity but lacking a signal sequence, a prepro domain, a dockerin domain, and/or a carbohydrate binding module (CBM),   wherein optionally the carbohydrate binding module (CBM) comprises, or consists of, a xylan binding module, a cellulose binding module, a lignin binding module, a xylose binding module, a mannanse binding module, a xyloglucan-specific module and/or a arabinofuranosidase binding module;   (e) the polypeptide of (a), (b), (c), or (d) having a xylanase, a mannanase and/or a glucanase activity further comprising a heterologous sequence;   (f) the polypeptide of (e), wherein the heterologous sequence comprises, or consists of: (i) a heterologous signal sequence, a heterologous carbohydrate binding module, a heterologous dockerin domain, a heterologous catalytic domain (CD), or a combination thereof; (ii) the sequence of (i), wherein the heterologous signal sequence, carbohydrate binding module or catalytic domain (CD) is derived from a heterologous lignocellulosic enzyme; and/or, (iii) a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme;   (g) the polypeptide of (f), wherein the heterologous carbohydrate binding module (CBM) comprises, or consists of, a xylan binding module, a cellulose binding module, a lignin binding module, a xylose binding module, a mannan binding module, a xyloglucan-specific module and/or a arabinofuranosidase binding module;   (h) polypeptide of (f), wherein the heterologous sequence targets the encoded protein to a vacuole, the endoplasmic reticulum, a chloroplast or a starch granule;   (i) the polypeptide of (a), (b), (c), (d), (e), (f), (g), or (h), wherein
 (A) the xylanase activity comprises catalyzing hydrolysis of internal β-1,4-xylosidic linkages; comprises an endo-1,4-beta-xylanase activity; comprises hydrolyzing a xylan or an arabinoxylan to produce a smaller molecular weight xylose and xylo-oligomer; comprises hydrolyzing a polysaccharide comprising a 1,4-β-glycoside-linked D-xylopyranose; comprises hydrolyzing a cellulose or a hemicellulose; comprises hydrolyzing a cellulose or a hemicellulose in a wood, wood product, paper pulp, paper product or paper waste; comprises catalyzing hydrolysis of a xylan or an arabinoxylan in a feed or a food product; or, comprises catalyzing hydrolysis of a xylan or an arabinoxylan in a microbial cell or a plant cell, wherein optionally the xylan or arabinoxylan comprises a water soluble arabinoxylan, and optionally the water soluble xylan or arabinoxylan comprises a dough or a bread product, wherein optionally the feed or food product comprises a cereal-based animal feed, a wort or a beer, a milk or a milk product, a fruit or a vegetable; 
 (B) the glucanase activity comprises an endoglucanase activity, e.g., endo-1,4- and/or 1,3-beta-D-glucan 4-glucano hydrolase activity; catalyzing hydrolysis of 1,4-beta-D-glycosidic linkages; an endo-1,4- and/or 1,3-beta-endoglucanase activity or endo-β-1,4-glucanase activity; an endo-1,4-beta-D-glucan 4-glucano hydrolase activity; catalyzing the hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives, carboxy methyl cellulose and/or hydroxy ethyl cellulose, lichenin, beta-1,4 bonds in mixed beta-1,3 glucans, cereal beta-D-glucans and/or other plant material containing cellulosic parts; hydrolyzing a glucan, a beta-glucan or a polysaccharide to produce a smaller molecular weight polysaccharide or oligomer; or, 
 (C) the mannanase activity comprises a endo-1,4-beta-D-mannanase activity, or catalyzing the hydrolysis of a beta-1,4-mannan or an unsubstituted linear beta-1,4-mannan, or 
   (j) the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), or (i), wherein optionally the polypeptide comprises at least one glycosylation site or further comprises a polysaccharide, wherein optionally the glycosylation is an N-linked glycosylation, and optionally the polypeptide is glycosylated after being expressed in a  P. pastoris  or a  S. pombe.      
     
     
         135 . An isolated, synthetic or recombinant polypeptide having xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide has a sequence comprising:
 (a) a sequence modification of the sequence of SEQ ID NO:384, wherein the sequence modification comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or all of the following changes:
 the amino acid at equivalent of the threonine at residue 4 of SEQ ID NO:384 is leucine, 
 the amino acid at the equivalent of the serine at residue 9 of SEQ ID NO:384 is proline, 
 the amino acid at the equivalent of the glutamine at residue 10 of SEQ ID NO:384 is serine, 
 the amino acid at the equivalent of the threonine at residue 13 of SEQ ID NO:384 is phenylalanine, 
 the amino acid at the equivalent of the threonine at residue 13 of SEQ ID NO:384 is tyrosine, 
 the amino acid at the equivalent of the threonine at residue 13 of SEQ ID NO:384 is isoleucine, 
 the amino acid at the equivalent of the threonine at residue 13 of SEQ ID NO:384 is tryptophan, 
 the amino acid at the equivalent of the asparagine at residue 14 of SEQ ID NO:384 is histidine, 
 the amino acid at the equivalent of the tyrosine at residue 18 of SEQ ID NO:384 is phenylalanine, 
 the amino acid at the equivalent of the serine at residue 25 of SEQ ID NO:384 is glutamic acid, 
 the amino acid at the equivalent of the serine at residue 25 of SEQ ID NO:384 is proline, 
 the amino acid at the equivalent of the asparagine at residue 30 of SEQ ID NO:384 is valine, 
 the amino acid at the equivalent of the glutamine at residue 34 of SEQ ID NO:384 is cysteine, 
 the amino acid at the equivalent of the glutamine at residue 34 of SEQ ID NO:384 is histidine, 
 the amino acid at the equivalent of the glutamine at residue 34 of SEQ ID NO:384 is leucine, 
 the amino acid at the equivalent of the serine at residue 35 of SEQ ID NO:384 is glutamic acid, 
 the amino acid at the equivalent of the serine at residue 35 of SEQ ID NO:384 is aspartic acid, 
 the amino acid at the equivalent of the serine at residue 71 of SEQ ID NO:384 is threonine, 
 the amino acid at the equivalent of the serine at residue 71 of SEQ ID NO:384 is cysteine, or 
 the amino acid at the equivalent of the serine at residue 194 of SEQ ID NO:384 is histidine; or 
   (b) one or more of the following changes to the amino acid sequence of SEQ ID NO:384:
 the threonine at amino acid position 4 is leucine, 
 the serine at amino acid position 9 is proline, 
 the glutamine at amino acid position 10 is serine, 
 the threonine at amino acid position 13 is phenylalanine, 
 the threonine at amino acid position 13 is tyrosine, 
 the threonine at amino acid position 13 is isoleucine, 
 the threonine at amino acid position 13 is tryptophan, 
 the asparagine at amino acid position 14 is histidine, 
 the tyrosine at amino acid position 18 is phenylalanine, 
 the serine at amino acid position 25 is glutamic acid, 
 the serine at amino acid position 25 is proline, 
 the asparagine at amino acid position 30 is valine, 
 the glutamine at amino acid position 34 is cysteine, 
 the glutamine at amino acid position 34 is histidine, 
 the glutamine at amino acid position 34 is leucine, 
 the serine at amino acid position 35 is glutamic acid, 
 the serine at amino acid position 35 is aspartic acid, 
 the serine at amino acid position 71 is threonine, 
 the serine at amino acid position 71 is cysteine, or 
 the serine at amino acid position 194 is histidine. 
   
     
     
         136 . A composition comprising a polypeptide of any of  claim 134  or  135  wherein optionally the composition is a pharmaceutical composition, a detergent composition, a contact lens solution, a waste treatment composition, a bar or liquid soap, or a chewing gum, lozenge or candy,
 wherein optionally the composition is an alcohol, wherein optionally the alcohol is ethanol, 
 wherein optionally the composition is a paper, paper waste, recycled paper product, newspaper, paper pulp, wood, wood product, wood waste, wood pulp, Kraft pulp, lignocellulose pulp, textile, fabric, yarn or a cloth, 
 wherein optionally the composition is a beverage, a food, a feed or a nutritional supplement, 
 wherein optionally the food is dough or bread, 
 wherein optionally the beverage, food, feed, or nutritional supplement is for an animal. 
 
     
     
         137 . A method of generating a variant of a polypeptide having a xylanase, a mannanase and/or a glucanase activity, comprising the steps of:
 (a) providing a template polypeptide comprising a sequence as set forth in any of  claim 134  or  135 ; and   (b) modifying, deleting or adding one or more amino acids in the template polypeptide, or a combination thereof, to generate a variant of the template polypeptide;   wherein optionally the modifications, additions or deletions are introduced by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a combination thereof, or, the modifications, additions or deletions are introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof;   and wherein optionally the method is iteratively repeated until a xylanase, a mannanase and/or a glucanase having an altered or different activity or an altered or different stability from that of the template polypeptide is produced,   wherein optionally the variant xylanase, a mannanase and/or a glucanase polypeptide is thermotolerant, and retains some activity after being exposed to an elevated temperature, or, optionally the variant xylanase, a mannanase and/or a glucanase polypeptide has increased glycosylation as compared to the xylanase, a mannanase and/or a glucanase of the template polypeptide, or optionally the variant xylanase, a mannanase and/or a glucanase polypeptide has a xylanase, a mannanase and/or a glucanase activity under a high temperature, wherein the xylanase, a mannanase and/or a glucanase of the template polypeptide is not active under the high temperature,   wherein optionally the method is iteratively repeated until a xylanase, a mannanase and/or a glucanase coding sequence having an altered codon usage from that of the template polypeptide is produced,   wherein optionally the method is iteratively repeated until a xylanase, a mannanase and/or a glucanase gene having higher or lower level of message expression or stability from that of the template polypeptide is produced.   
     
     
         138 . A chimeric polypeptide comprising:
 (a) a polypeptide or peptide comprising a sequence as set forth in any of  claim 134  or  135 , or a subsequence thereof, and   (b) (i) a sequence as set forth in residues 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46, 1 to 47, 1 to 48, 1 to 49 or 1 to 50, of a polypeptide as set forth in any of  claim 134  or  135 ; (ii) a sequence as set forth in Table 4;   (iii) a heterologous carbohydrate-binding module (CBM); wherein optionally the CBM comprises a CBM3a, CBM3b, CBM4, CBM6, CBM22 or X14, a carbohydrate-binding subsequence of a sequence as set forth in any of  claim 134  or  135 , or a carbohydrate-binding subsequence comprising a X14 as set forth in Table 9.   
     
     
         139 . A method for hydrolyzing, liquefying, breaking up or disrupting a xylan-comprising composition comprising the following steps:
 (a) providing a polypeptide having a xylanase, a mannanase and/or a glucanase activity,   wherein the polypeptide comprises a sequence as set forth in any of  claim 134  or  135 , or enzymatically active fragments thereof;   (b) providing a composition comprising a xylan; and   (c) contacting the polypeptide of step (a) with the composition of step (b) under conditions wherein the xylanase, a mannanase and/or a glucanase hydrolyzes, liquefies, breaks up or disrupts the xylan-comprising composition,   wherein optionally the composition comprises a plant cell, a bacterial cell, a yeast cell, an insect cell, or an animal cell.   
     
     
         140 . A method of food, feed or beverage production comprising-use of at least one polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide comprises a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof. 
     
     
         141 . A method for utilizing a xylanase, a mannanase and/or a glucanase as a food or as a nutritional supplement in an animal diet, the method comprising:
 preparing a food or a nutritional supplement containing a xylanase, a mannanase and/or a glucanase enzyme comprising at least thirty contiguous amino acids of a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide comprises a sequence as set forth in any of  claim 134  or  135 , or enzymatically active fragments thereof; and   administering the food or nutritional supplement to an animal,   wherein optionally the animal is a human or non-human, and optionally the animal is a ruminant or a monogastric animal.   
     
     
         142 . A method for eliminating or protecting animals from a microorganism comprising a xylan comprising administering a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide comprises a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof, wherein optionally the microorganism is a bacterium or a salmonellae. 
     
     
         143 . A method for bleaching, decoloring or deinking of a composition, comprising:
 contacting the composition with a xylanase, a mannanase and/or a glucanase, wherein the xylanase, a mannanase and/or a glucanase has a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof, wherein optionally the composition is a paper, paper waste, recycled paper product, newspaper, paper pulp, wood, wood product, wood waste, wood pulp, Kraft pulp, lignocellulose pulp, textile, fabric, yarn or a cloth;   wherein optionally the method further comprises a bleaching agent, wherein optionally the bleaching agent comprises oxygen or hydrogen peroxide;   wherein optionally the method further comprises a filtration step, and optionally a filtrate is generated, and optionally the method further comprises recycling of the filtrate, and optionally fines are collected from the filtrate;   and optionally the method further comprises a mixing step, and optionally the xylanase, a mannanase and/or a glucanase is added at multiple time points or step-wise in the method, and optionally the xylanase, a mannanase and/or a glucanase is added at different times or step-wise in the bleaching process;   and optionally the method further comprises addition of additional enzymes, and optionally the additional enzymes are added at multiple time points or step-wise in the method; and optionally the method further comprises a pre-washing step or a pretreatment step, and optionally the method comprises pre-washing step or a pretreatment with the xylanase, a mannanase and/or a glucanase;   and optionally the method comprises high temperature and high pH conditions.   
     
     
         144 . A method for reducing, releasing or solubilizing lignin in a composition comprising:
 contacting the composition with a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide has a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof, wherein optionally the composition is a paper, paper waste, recycled paper product, newspaper, paper pulp, wood, wood product, wood waste, wood pulp, Kraft pulp, lignocellulose pulp, textile, fabric, yarn or a cloth;   wherein optionally the method further comprises a filtration step, and optionally a filtrate is generated;   and optionally the method further comprises recycling of the filtrate, and optionally fines are collected from the filtrate;   and optionally the method further comprises a mixing step, and optionally the xylanase, a mannanase and/or a glucanase is added at multiple time points or step-wise in the method, and optionally the method further comprises a bleaching process, and optionally the xylanase, a mannanase and/or a glucanase is added at different times or step-wise in the bleaching process; and optionally the method further comprises addition of additional enzymes, and optionally the additional enzymes are added at multiple time points or step-wise in the method;   and optionally the method further comprises a pre-washing step or a pretreatment step, and optionally the method comprises pre-washing step or a pretreatment with the xylanase, a mannanase and/or a glucanase;   and optionally the method comprises high temperature and high pH conditions;   and optionally after the method, the pulp has a consistency of about 10%.   
     
     
         145 . A method for treating a wood, a wood pulp, a Kraft pulp, a paper product, a paper or a paper pulp, the method comprising the following steps:
 (a) providing at least one polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide retains xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide comprises a xylanase, a mannanase and/or a glucanase having a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof;   (b) providing a wood, a wood pulp, a Kraft pulp, a paper, a paper product or a paper pulp; and   (c) contacting the wood, wood pulp, Kraft pulp, paper, paper product or paper pulp with the polypeptide of step (a), wherein the polypeptide catalyzes hydrolysis of compounds in the wood, wood pulp, Kraft pulp, paper, paper product or paper pulp, and wherein optionally the wood, wood pulp, Kraft pulp, paper, paper product or paper pulp comprises a softwood and hardwood, or the wood, wood pulp, Kraft pulp, paper or paper pulp is derived from a softwood and hardwood;   and wherein optionally after the treatment the pulp has a consistency of at least about 10%, or at least about 32%;   wherein optionally the method further comprises a filtration step, and optionally a filtrate is generated, and optionally the method further comprises recycling of the filtrate, and optionally fines are collected from the filtrate;   and optionally the method further comprises a mixing step, and optionally the xylanase, a mannanase and/or a glucanase is added at multiple time points or step-wise in the method, and optionally the method further comprises a bleaching process, and optionally the xylanase, a mannanase and/or a glucanase is added at different times or step-wise in the bleaching process; and optionally the method further comprises addition of additional enzymes, and optionally the additional enzymes are added at multiple time points or step-wise in the method;   and optionally the method further comprises a pre-washing step or a pretreatment step,   and optionally the method comprises pre-washing step or a pretreatment with the xylanase, a mannanase and/or a glucanase.   
     
     
         146 . A method for making an alcohol comprising (a) contacting a composition with a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide has a sequence as set forth in any of  claim 134  or  135 -, or an enzymatically active fragment thereof,
 wherein optionally the composition comprises a saccharide, a hemicellulose, a cellulose, a lignin, or a combination thereof; 
 and optionally the alcohol comprises an ethanol; 
 and optionally, the method comprises high temperature and basic pH conditions, 
 and optionally the method further comprises a filtration step, and optionally a filtrate is generated; 
 and optionally the method further comprises recycling of the filtrate, and optionally fines are collected from the filtrate; 
 and optionally the method further comprises a mixing step, and optionally the xylanase, a mannanase and/or a glucanase is added at multiple time points or step-wise in the method, and optionally the method further comprises a bleaching process, and optionally the xylanase, a mannanase and/or a glucanase is added at different times or step-wise in the bleaching process; and optionally the method further comprises addition of additional enzymes, and optionally the additional enzymes are added at multiple time points or step-wise in the method; 
 and optionally the method further comprises a pre-washing step or a pretreatment step, and optionally the method comprises pre-washing step or a pretreatment with the xylanase, a mannanase and/or a glucanase. 
 
     
     
         147 . A method for designing a chimeric glycosidase, xylanase, a mannanase and/or a glucanase having a new carbohydrate-binding specificity or an enhanced carbohydrate-binding specificity, comprising inserting a heterologous or an additional endogenous carbohydrate-binding module (CBM) into a glycosidase, wherein the CBM comprises a carbohydrate-binding subsequence of a sequence as set forth in any of  claim 134  or  135 , or a carbohydrate-binding subsequence comprising a X14 as set forth in Table 9. 
     
     
         148 . An enzyme mixture or cocktail, comprising:
 (a) at least one enzyme of any of any of  claim 134  or  135 , and one or more other enzyme(s); or   (b) the mixture or cocktail of (a), wherein the one or more other enzyme(s) is another xylanase, a mannanase and/or a glucanase, cellulases, lipases, esterases, proteases, or endoglycosidases, endo-beta.-1,4-glucanases, beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases, catalases, laccases, amylases, glucoamylases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases, mannanases, xyloglucanases, xylanase, a mannanase and/or a glucanases, pectin acetyl esterases, rhamnogalacturonan acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, pectin methylesterases, cellobiohydrolases and/or transglutaminases.   
     
     
         149 . A process for hydrolyzing a hemicellulose, cellulose, lignin or saccharide in any organic compound, plant or wood or wood product or wood pulp or byproduct, wood waste, paper, paper pulp, paper product or paper waste or byproduct, comprising:
 using an enzyme mixture or cocktail of  claim 148 , the polypeptide of  claim 134  or  135 , or an enzymatically active fragment thereof,   wherein optionally the method further comprises a filtration step, and optionally a filtrate is generated;   and optionally the method further comprises recycling of the filtrate (to catch fines);   and optionally the method further comprises a mixing step, and optionally the polypeptide having a xylanase, a mannanase and/or a glucanase activity is added at multiple time points or step-wise in the method;   and optionally the method further comprises a bleaching process, and optionally the polypeptide having a xylanase, a mannanase and/or a glucanase activity is added at different times or step-wise in the bleaching process;   and optionally the method further comprises addition of additional enzymes, and optionally the additional enzymes are added at multiple time points or step-wise in the method; and optionally the method further comprises a pre-washing step or a pretreatment step, and optionally the method comprises pre-washing step or a pretreatment with the polypeptide having a xylanase, a mannanase and/or a glucanase activity.   
     
     
         150 . A method for increasing performance of a xylanase at a high (alkaline) pH, comprising:
 (a) removing amino acid residues “EGG” (or the equivalent) near or at the C′ terminal end of a xylanase sequence;   (b) the method of (a), wherein the “EGG” (or the equivalent) is removed just (immediately) after the glycosyl hydrolase domain of the xylanase to be modified; or   (c) the method of (a) or (b), wherein the xylanase comprises a polypeptide of any of  claim 134  or  135 .   
     
     
         151 . A method for reducing the amount of bleaching chemical in a wood, wood pulp, wood product, Kraft pulp, paper, paper pulp, paper product, or recycled paper process, comprising:
 (a) providing a polypeptide having xylanase, mannanase and/or glucanase activity, wherein the polypeptide comprises the polypeptide of  claim 134  or  135 , or an enzymatically active fragment thereof;   (b) providing a wood, wood pulp, wood product, Kraft pulp, paper, paper pulp, paper product, or recycled paper; and   (c) contacting the wood, wood pulp, wood product, Kraft pulp, paper, paper pulp, paper product, or recycled paper with the polypeptide of (a),   wherein optionally the bleaching chemical comprises a chlorine, a chlorine dioxide, a caustic, a peroxide, or any combination thereof,   wherein optionally the method further comprises a filtration step, and optionally a filtrate is generated;   and optionally the method further comprises recycling of the filtrate, and optionally fines are collected from the filtrate;   and optionally the method further comprises a mixing step, and optionally the xylanase, a mannanase and/or a glucanase is added at multiple time points or step-wise in the method;   and optionally the method further comprises addition of additional enzymes, and optionally the additional enzymes are added at multiple time points or step-wise in the method;   and optionally the method further comprises a pre-washing step or a pretreatment step, and optionally the method comprises pre-washing step or a pretreatment with the xylanase, a mannanase and/or a glucanase;   and optionally conditions for treatment after an oxygen delignification step (a post-O 2  pulp) with the xylanase enzyme comprise: pH of between about 6 to 7, enzyme dose of about 0.3 units/g, treatment time of between about 20 to 25 minutes;   and optionally the method comprises a pretreatment of post-O 2  spruce/pine/fir (SPF) pulp with about 2 units/g of xylanase to reduce subsequent ClO 2  use increase brightness;   and optionally the method comprises a pretreatment of pre-O 2  brownstock SPF with about 0.5 units/g of xylanase to reduce subsequent ClO 2  use increase brightness;   and optionally the method comprises a pretreatment of pre-O 2  Aspen pulp with about 0.5 units/g of xylanase to reduce subsequent ClO 2  use increase brightness;   and optionally the method comprises a pretreatment of pre-O 2  Douglas Fir/Hemlock pulp with about 0.5 units/g of xylanase to reduce subsequent ClO 2  use increase brightness.   
     
     
         152 . A method for board manufacturing comprising contacting a bleached or an unbleached pulp or recycled paper pulp with a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein optionally the polypeptide has a sequence as set forth in any of  claim 134  or  135  or an enzymatically active fragment thereof. 
     
     
         153 . A method for lowering alkali in cooking or to decrease cooking in board manufacturing comprising contacting a bleached or an unbleached pulp or recycled paper pulp with a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein optionally the polypeptide has a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof. 
     
     
         154 . A method to increase Kappa number during cooking or to increase pulp strength in board manufacturing comprising contacting a bleached or an unbleached pulp or recycled paper with a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein optionally the polypeptide has a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof. 
     
     
         155 . A method for making a sugar, comprising:
 (a) contacting a polysaccharide-comprising composition with a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide has a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof, wherein the contacting results in the generation of a sugar;   (b) the method of (a), wherein the polysaccharide-comprising composition comprises a saccharide, a hemicellulose, a cellulose, a lignin or a combination thereof;   (c), the method of (a) or (b), comprising use of an enzyme mixture or cocktail of  claim 148 ; or   (d) the method of (a), (b) or (c), further comprising fermenting the sugar to produce an alcohol.   
     
     
         156 . A process for enzymatic hydrolysis of a polysaccharide, comprising:
 (a) contacting a polysaccharide-comprising composition with a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide has a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof, wherein the contacting results in the hydrolysis of the polysaccharide to produce a sugar;   (b) the method of (a), wherein the polysaccharide-comprising composition comprises an organic compound, a plant or a wood or a wood product or byproduct, a wood waste, a paper pulp, a paper product or a paper waste or a byproduct;   (c), the method of (a) or (b), comprising use of an enzyme mixture or cocktail of  claim 148 ;   (d) the method of (a), (b) or (c), wherein the hydrolysis of the polysaccharide results in the generation of a monomeric sugar; or   (e) the method of (a), (b), (c) or (d), further comprising fermenting the carbohydrate or sugar to produce an alcohol.   
     
     
         157 . A fermentation process (a process for converting a carbohydrate into an alcohol), comprising:
 (a) (i) contacting a carbohydrate-comprising composition with a polypeptide having a xylanase, a mannanase and/or a glucanase activity, wherein the polypeptide has a sequence as set forth in any of  claim 134  or  135 , or an enzymatically active fragment thereof, wherein the contacting results in the enzymatic hydrolysis of the carbohydrate; (ii) fermenting the carbohydrate generated in step (i) to produce an alcohol;   (b) the method of (a), wherein the carbohydrate-comprising composition comprises an organic compound, a plant or a wood or a wood product or byproduct, a wood waste, a paper pulp, a paper product or a paper waste or a byproduct;   (c), the method of (a) or (b), comprising use of an enzyme mixture or cocktail of  claim 148 ; or   (d) the method of (a), (b) or (c), wherein the fermentation in step (a)(ii) (the conversion of the carbohydrate into the alcohol) results in the generation of ethanol.