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 . An isolated, synthetic or recombinant nucleic acid comprising a nucleic acid encoding a polypeptide having a xylanase activity, wherein the nucleic acid comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:1 and has at least one mutation selected from (a) to (m), and wherein the said polypeptide shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at the same enzyme loading, and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at a reduced enzyme loading:
 (a) the nucleic acid codon encoding amino acid residue 38 of SEQ ID NO: 2 is changed from CGT to CAC, and amino acid residue 38 Arginine, Arg (or “R”) is changed to Histidine, H is (or “H”);   (b) the nucleic acid codon encoding amino acid residue 33 of SEQ ID NO: 2 is changed from CTG to GCG, and amino acid residue 33 Leucine, Leu (or “L”) is changed to Alanine, Ala (or “A”);   (c) the nucleic acid codon encoding amino acid residue 73 of SEQ ID NO: 2 is changed from GGC to TAC, and amino acid residue 73 Glycine, Gly (or “G”) is changed to Tyrosine, “Tyr” (or “Y”);   (d) the nucleic acid codon encoding amino acid residue 63 of SEQ ID NO: 2 is changed from ATG to GTC, and amino acid residue 63 Isoleucine, Ile (or “I”) is changed to Valine, “Val” (or “V”);   (e) the nucleic acid codon encoding amino acid residue 188 of SEQ ID NO: 2 is changed from AGC to GAG, and amino acid residue 188 Serine, Ser (or “S”) is changed to Glutamate, Glu (or “E”);   (f) the nucleic acid codon encoding amino acid residue 21 of SEQ ID NO: 2 is changed from TTC to TAC, and the amino acid residue 21 Phenylalanine, Phe (or “F”) is changed to Tyrosine, “Tyr” (or “Y”);   (g) the nucleic acid codon encoding amino acid residue 125 of SEQ ID NO: 2 is changed from CAG to TAC, and amino acid residue 125 Glutamine, Gln (or “Q”) is changed to Tyrosine, “Tyr” (or “Y”);   (h) the nucleic acid codon encoding amino acid residue 4 of SEQ ID NO: 2 is changed from ACC to CGC, and amino acid residue 4 Threonine, or “Thr” (or “T”) is changed to Arginine, Arg (or “R”);   (i) the nucleic acid codon encoding amino acid residue 73 of SEQ ID NO: 2 is changed from GGC to GAG, and amino acid residue 73 Glycine, Gly (or “G”) is changed to Glutamate, Glu (or “E”);   (j) the nucleic acid codon encoding amino acid residue 150 of SEQ ID NO: 2 is changed from GTA to GCC, and the amino acid residue 150 Valine, Val (or “V”) is changed to Alanine, Ala (or “A”);   (k) the nucleic acid codon encoding amino acid residue 17 of SEQ ID NO: 2 is changed from TTC to GTC, and amino acid residue 17 Phenylalanine, Phe (or “F”) is changed to Valine, “Val” (or “V”);   (i) the nucleic acid codon encoding amino acid residue 4 of SEQ ID NO: 2 is changed from ACC to CAC, and amino acid residue 4 Threonine, or “Thr” (or “T”) is changed to Histidine, H is (or “H”); and   (m) the nucleic acid codon encoding amino acid residue 9 of SEQ ID NO: 2 is changed from CCC to GAC, and the amino acid residue 9 Proline, Pro (or “P”) is changed to Aspartate, Asp (or “D”);   
     
     
         2 . The isolated, synthetic or recombinant nucleic acid comprising a nucleic acid encoding a polypeptide having a xylanase activity of  claim 1 , wherein the nucleic acid has one or more further nucleotide residue changes selected from:
 a) the nucleic acid codon encoding amino acid residue 4 of SEQ ID NO: 2 is changed from ACC to AAC, and amino acid residue 4 is changed from T to N;   b) the nucleic acid codon encoding amino acid residue 4 of SEQ ID NO: 2 is changed from ACC to CGC, and amino acid residue 4 is changed from T to R;   c) the nucleic acid codon encoding amino acid residue 4 of SEQ ID NO: 2 is changed from ACC to CAC, and amino acid residue 4 is changed from T to H;   d) the nucleic acid codon encoding amino acid residue 9 of SEQ ID NO: 2 is changed from CCC to GAC, and amino acid residue 9 is changed from P to D;   e) the nucleic acid codon encoding amino acid residue 17 of SEQ ID NO: 2 is changed from TTC to GTC, and amino acid residue 17 is changed from F to V;   f) the nucleic acid codon encoding amino acid residue 21 of SEQ ID NO: 2 is changed from TTC to TAC, and amino acid residue 21 is changed from F to Y;   g) the nucleic acid codon encoding amino acid residue 33 of SEQ ID NO: 2 is changed from CTG to GCG, and amino acid residue 33 is changed from L to A;   h) the nucleic acid codon encoding amino acid residue 38 of SEQ ID NO: 2 is changed from CGT to CAC, and amino acid residue 38 is changed from R to H;   i) the nucleic acid codon encoding amino acid residue 44 of SEQ ID NO: 2 is changed from AGC to ACG, and amino acid residue 44 is changed from S to T;   j) the nucleic acid codon encoding amino acid residue 63 of SEQ ID NO: 2 is changed from ATC to GTC, and amino acid residue 63 is changed from I to V;   k) the nucleic acid codon encoding amino acid residue 73 of SEQ ID NO: 2 is changed from GGC to TAC, and amino acid residue 73 is changed from G to Y;   l) the nucleic acid codon encoding amino acid residue 73 of SEQ ID NO: 2 is changed from GGC to GAG, and amino acid residue 73 is changed from G to E;   m) the nucleic acid codon encoding amino acid residue 73 of SEQ ID NO: 2 is changed from GGC to GTC, and amino acid residue 73 is changed from G to V;   n) the nucleic acid codon encoding amino acid residue 108 of SEQ ID NO: 2 is changed from TTC to AAG, and amino acid residue 108 is changed from F to K;   o) the nucleic acid codon encoding amino acid residue 125 of SEQ ID NO: 2 is changed from CAG to TAC, and amino acid residue 125 is changed from Q to Y;   p) the nucleic acid codon encoding amino acid residue 150 of SEQ ID NO: 2 is changed from GTA to GCC, and amino acid residue 150 is changed from V to A;   q) the nucleic acid codon encoding amino acid residue 188 of SEQ ID NO: 2 is changed from AGC to GAG, and amino acid residue 188 is changed from S to E; and   r) the nucleic acid codon encoding amino acid residue 189 of SEQ ID NO: 2 is changed from TCC to CAG, and amino acid residue 189 is changed from S to Q;   
       wherein the said polypeptide shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at the same enzyme loading, and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at a reduced enzyme loading 
     
     
         3 . The nucleic acid of  claim 1 , wherein the nucleic acid has one or more nucleic acid encodes at least one polypeptide having at least one further amino acid residue change (or the equivalent thereof) selected from:
 a) the amino acid residue 4 is changed from T to N;   b) the amino acid residue 4 is changed from T to R;   c) the amino acid residue 4 is changed from T to H;   d) the amino acid residue 9 is changed from P to D;   e) the amino acid residue 17 is changed from F to V;   f) the amino acid residue 21 is changed from F to Y;   g) the amino acid residue 33 is changed from L to A;   h) the amino acid residue 38 is changed from R to H;   i) the amino acid residue 44 is changed from S to T;   j) the amino acid residue 63 is changed from I to V;   k) the amino acid residue 73 is changed from G to Y;   l) the amino acid residue 73 is changed from G to E;   m) the amino acid residue 73 is changed from G to V;   n) the amino acid residue 108 is changed from F to K;   o) the amino acid residue 125 is changed from Q to Y;   p) the amino acid residue 150 is changed from V to A;   q) the amino acid residue 188 is changed from S to E; and   r) the amino acid residue 189 is changed from S to Q;   
       wherein the nucleic acid of  claim 1  or  2  comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1 and it encodes a polypeptide which shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at the same enzyme loading, and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at a reduced enzyme loading. 
     
     
         4 . The nucleic acid of  claim 2 , wherein the nucleic acid further comprises one or more nucleotide changes such that the nucleic acid encodes a polypeptide having at least one conservative amino acid substitution and retains its xylanase activity; and, optionally, wherein the at least one conservative amino acid substitution comprises substituting an amino acid with another amino acid of like characteristics; or, a conservative substitution comprises a 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; and wherein the nucleic acid comprises a sequence having at least 85% sequence identity to SEQ ID NO: 1 and it encodes a polypeptide which shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at the same enzyme loading, and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at a reduced enzyme loading. 
     
     
         5 . The nucleic acid of  claim 2  wherein comprises a heterologous nucleic acid sequence or further comprises a heterologous nucleic acid sequence encoding a heterologous polypeptide sequence. 
     
     
         6 . The nucleic acid of  claim 5 , wherein the heterologous polypeptide sequence comprises a heterologous signal sequence, a heterologous carbohydrate binding module (CBM), a heterologous dockerin domain, a heterologous catalytic domain (CD), or a combination thereof. 
     
     
         7 . The nucleic acid of  claim 6 , wherein the heterologous signal sequence, carbohydrate binding module or catalytic domain (CD) is derived from a heterologous enzyme; or a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme. 
     
     
         8 . The nucleic acid of  claim 6 , wherein the heterologous carbohydrate binding module (CBM) comprises 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 an arabinofuranosidase binding module. 
     
     
         9 . The nucleic acid of  claim 6 , the heterologous signal sequence targets the encoded protein to a vacuole, the endoplasmic reticulum, a chloroplast or a starch granule. 
     
     
         10 . A nucleic acid sequence which is fully (completely) complementary to the sequence of  claim 1 . 
     
     
         11 . A vector comprising the sequence of  claim 1 , wherein the vector is a viral vector comprising an adenovirus vector, a retroviral vector or an adeno-associated viral vector. 
     
     
         12 . A transformed cell comprising a nucleic acid of  claim 1  wherein the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell. 
     
     
         13 . A transgenic plant, plant part or plant seed, or a transgenic non-human animal, comprising a nucleic acid of  claim 1 , wherein the plant is a corn plant, a sorghum plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant, a grass, a cotton plant, a cottonseed plant, a palm, a sesame plant, a peanut plant, a sunflower plant or a tobacco plant. 
     
     
         14 . An isolated, synthetic or recombinant polypeptide or peptide having a xylanase activity, wherein the polypeptide or peptide comprises a sequence having at least 95%, 96%, 97%, 98%, 99%, or more, or complete (100%), sequence identity to SEQ ID NO:2 having at least one mutation selected from (a) to (m) and wherein the said polypeptide with xylanase activity shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at the same enzyme loading, and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at a reduced enzyme loading:
 (a) the amino acid residue 38 Arginine, Arg (or “R”) is changed to Histidine, H is (or “H”) ;      (b) the amino acid residue 33 Leucine, Leu (or “L”) is changed to Alanine, Ala (or “A”);   (c) the amino acid residue 73 Glycine, Gly (or “G”) is changed to Tyrosine, “Tyr” (or “Y”);   (d) the amino acid residue 63 Isoleucine, Ile (or “I”) is changed to Valine, “Val” (or “V”);   (e) the amino acid residue 188 Serine, Ser (or “S”) is changed to Glutamate, Glu (or “E”);   (f) the amino acid residue 21 Phenylalanine, Phe (or “F”) is changed to Tyrosine, “Tyr” (or “Y”);   (g) the amino acid residue 125 Glutamine, Gln (or “Q”) is changed to Tyrosine, “Tyr” (or “Y”);   (h) the amino acid residue 4 Threonine, or “Thr” (or “T”) is changed to Arginine, Arg (or “R”);   (i) the amino acid residue 73 Glycine, Gly (or “G”) is changed to Glutamate, Glu (or “E”);   (j) the amino acid residue 150 Valine, Val (or “V”) is changed to Alanine, Ala (or “A”);   (k) the amino acid residue 17 Phenylalanine, Phe (or “F”) is changed to Valine, “Val” (or “V”);   (l) the amino acid residue 4 Threonine, or “Thr” (or “T”) is changed to Histidine, H is (or “H”); and   (m) the amino acid residue 9 Proline, Pro (or “P”) is changed to Aspartate, Asp (or “D”).   
     
     
         15 . The isolated, synthetic or recombinant polypeptide or peptide of  claim 14 , wherein the polypeptide or peptide has at least one of the following further amino acid residue changes:
 a) the amino acid residue 4 is changed from T to N;   b) the amino acid residue 4 is changed from T to R;   c) the amino acid residue 4 is changed from T to H;   d) the amino acid residue 9 is changed from P to D;   e) the amino acid residue 17 is changed from F to V;   f) the amino acid residue 21 is changed from F to Y;   g) the amino acid residue 33 is changed from L to A;   h) the amino acid residue 38 is changed from R to H;   i) the amino acid residue 44 is changed from S to T;   j) the amino acid residue 63 is changed from I to V;   k) the amino acid residue 73 is changed from G to Y;   l) the amino acid residue 73 is changed from G to E;   m) the amino acid residue 73 is changed from G to V;   n) the amino acid residue 108 is changed from F to K;   o) the amino acid residue 125 is changed from Q to Y;   p) the amino acid residue 150 is changed from V to A;   q) the amino acid residue 188 is changed from S to E; or   r) the amino acid residue 189 is changed from S to Q   
       and wherein the polypeptide or peptide comprises a sequence having at least 95% identity to SEQ ID NO: 2 and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at the same enzyme loading, and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at a reduced enzyme loading. 
     
     
         16 . The isolated, synthetic or recombinant polypeptide or peptide of  claim 14 , wherein the polypeptide further comprises at least one conservative amino acid substitution and retains its xylanase activity; wherein, optionally, the at least one conservative amino acid substitution comprises substituting an amino acid with another amino acid of like characteristics; or, a 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; wherein the polypeptide or peptide comprises a sequence having at least 95% identity to SEQ ID NO: 2 and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at the same enzyme loading, and shows an improved rate of xylose release when compared to assays with SEQ ID NO: 2, at a reduced enzyme loading. 
     
     
         17 . The isolated, synthetic or recombinant polypeptide or peptide of  claim 14 , wherein the polypeptide or peptide is encoded by the nucleic acid of  claim 1 . 
     
     
         18 . The isolated, synthetic or recombinant polypeptide or peptide of  claim 14 , wherein the polypeptide or peptide further comprises a heterologous amino or polypeptide or peptide sequence. 
     
     
         19 . The isolated, synthetic or recombinant polypeptide or peptide of  claim 18 , wherein the heterologous amino or polypeptide or peptide sequence comprises, a heterologous signal sequence, a heterologous carbohydrate binding module (CBM), a heterologous dockerin domain, a heterologous catalytic domain (CD), a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme, or a combination thereof. 
     
     
         20 . The isolated, synthetic or recombinant polypeptide or peptide of  claim 19 , wherein the heterologous signal sequence, carbohydrate binding module or catalytic domain (CD) is derived from a heterologous enzyme. 
     
     
         21 . The isolated, synthetic or recombinant polypeptide or peptide of  claim 19 , wherein the heterologous carbohydrate binding module (CBM) comprises 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 an arabinofuranosidase binding module, or a combination thereof. 
     
     
         22 . The isolated, synthetic or recombinant polypeptide or peptide of  claim 19 , wherein the heterologous signal sequence targets the encoded protein to a vacuole, the endoplasmic reticulum, a chloroplast or a starch granule. 
     
     
         23 . A protein preparation, or an immobilized polypeptide, comprising the polypeptide of  claim 14 , wherein the protein preparation comprises a liquid, a solid or a gel. 
     
     
         24 . An isolated, synthetic or recombinant antibody that specifically binds to the polypeptide of  claim 14 , wherein optionally the antibody is a monoclonal or a polyclonal antibody, or is a single chained antibody. 
     
     
         25 . An industrial process or a method for using a polypeptide having a xylanase activity, wherein the polypeptide comprises an amino acid sequence of  claim 14 , or the polypeptide is encoded by a nucleic acid of  claim 1 , or enzymatically active fragments thereof; comprising:
 (a) a method for hydrolyzing, liquefying, breaking up or disrupting a xylan-, cellulose- or hemicellulose-comprising composition comprising contacting the polypeptide or enzymatically active fragments thereof with a composition comprising a xylan, a cellulose or a hemicellulose under conditions wherein the polypeptide or enzymatically active fragments thereof hydrolyze, liquefy, break up or disrupt the xylan-, cellulose- or hemicellulose-comprising composition, wherein optionally the composition comprises a plant cell or a bacterial cell,   (b) a method for reducing the amount of lignin (delignification), or solubilizing a lignin, in a paper or paper product, a wood, wood pulp or wood product, or a wood or paper recycling composition, comprising contacting the paper or paper product, wood, wood pulp or wood product, or wood or paper recycling composition with the polypeptide or enzymatically active fragments thereof;   (c) a method for hydrolyzing celluloses, hemicelluloses or xylans in a biomass, a wood, wood product, paper pulp, paper product or paper waste comprising contacting the wood, wood product, paper pulp, paper product or paper waste with the polypeptide or enzymatically active fragments thereof;   (d) a method for enzymatic decoloring of paper, hemp or flax pulp comprising contacting the paper, hemp or flax pulp with the polypeptide or enzymatically active fragments thereof and a bleaching agent, wherein optionally the decoloring agent comprises oxygen or hydrogen peroxide under conditions suitable for enzymatic decoloring;   (e) a method for of decoloring a lignocellulose pulp comprising contacting the lignocellulose pulp with the polypeptide or enzymatically active fragments thereof under conditions suitable for enzymatic decoloring;   (f) a method for enzymatic deinking of paper, paper waste, paper recycled product, deinking toner from non-contact printed wastepaper or mixtures of non-contact and contact printed wastepaper, comprising contacting the paper, paper waste, paper recycled product, non-contact printed wastepaper or contact printed wastepaper with the polypeptide or enzymatically active fragments thereof, under conditions suitable for enzymatic deinking;   (g) a method for decoloring or deinking newspaper comprising contacting the newspaper with the polypeptide or enzymatically active fragments thereof under conditions suitable for enzymatic decoloring or deinking; or   (h) method for reducing lignin in a composition selected from a wood or wood product, a wood pulp, a Kraft pulp, a paper, a paper product or a paper pulp, comprising contacting the composition with the polypeptide or enzymatically active fragments thereof under conditions suitable for enzymatic reducing of the lignin.   
     
     
         26 . An enzyme mixture or cocktail comprising:
 (a) at least one polypeptide of  claim 14 , or a polypeptide encoded by a nucleic acid of  claim 1 , 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.   
     
     
         27 . A process for hydrolyzing xylans, celluloses or hemicelluloses in any organic compound, plant or wood or wood product or byproduct, wood waste, paper pulp, paper product or paper waste or byproduct comprising use of a polypeptide of  claim 14 , or a polypeptide encoded by a nucleic acid of  claim 1 , or an enzymatically active fragment thereof, or the enzyme mixture or cocktail of  claim 26 , or a combination thereof. 
     
     
         28 . A liquid composition comprising:
 (a) (i) an alcohol and (ii) a polypeptide of  claim 14 , or a polypeptide encoded by a nucleic acid of  claim 1 , or an enzymatically active fragment thereof, or the enzyme mixture or cocktail of  claim 26 , or a combination thereof;   (b) the liquid composition of (a), wherein the alcohol is or comprises ethanol, propanol, butanol and/or methanol; or   (c) the liquid composition of (a) or (b) comprising or contained in a fuels, a biofuel, a synthetic liquid or gas or a syngas.   
     
     
         29 . The enzyme mixture or cocktail of  claim 26 , comprising an Endo glucanase, an Oligomerase I (beta-glucosidase), a CBH1 (GH family 7), a CBH2 (GH family 6), a Xylanase (GH family 11), an Arabinofuranosidase, a Xylanase (GH family 10) and an Oligomerase II (beta-xylosidase), wherein at least one of these enzymes is a polypeptide of  claim 14 , or a polypeptide encoded by a nucleic acid of  claim 1 .