Novel fungal enzymes

Abstract

This invention relates to novel enzymes and novel methods for producing the same. More specifically this invention relates to a variety of fungal enzymes. Nucleic acid molecules encoding such enzymes, compositions, recombinant and genetically modified host cells, and methods of use are described. The invention also relates to a method to convert lignocellulosic biomass to fermentable sugars with enzymes that degrade the lignocellulosic material and novel combinations of enzymes, including those that provide a synergistic release of sugars from plant biomass. The invention also relates to a method to release cellular content by degradation of cell walls. The invention also relates to methods to use the novel enzymes and compositions of such enzymes in a variety of other processes, including washing of clothing, detergent processes, biorefining, deinking and biobleaching of paper and pulp, and treatment of waste streams.

What is claimed is: 
     
         1 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
 (a) a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 83, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 98, SEQ ID NO: 101, SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 113, SEQ ID NO: 116, SEQ ID NO: 119, SEQ ID NO: 122, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 131, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 140, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 158, SEQ ID NO: 161, SEQ ID NO: 164, SEQ ID NO: 167, SEQ ID NO: 170, SEQ ID NO: 173, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 182, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 194, SEQ ID NO: 197, SEQ ID NO: 200, SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 209, SEQ ID NO: 212, SEQ ID NO: 215, SEQ ID NO: 218, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 227, SEQ ID NO: 230, and SEQ ID NO: 233.   (b) a nucleic acid sequence encoding a fragment of a polypeptide specified in (a), wherein the fragment retains at least one biological activity said polypeptide; and   (c) a nucleic acid sequence encoding an amino acid sequence that is at least about 70% identical to a polypeptide specified in (a) and retains at least one biological activity of said polypeptide.   
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein said nucleic acid sequence comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 232, and SEQ ID NO: 234. 
     
     
         3 . A vector comprising the nucleic acid of  claim 1 . 
     
     
         4 . A vector comprising the nucleic acid of  claim 1 , wherein said nucleic acid is operably-linked to a promoter. 
     
     
         5 . A modified cell comprising the nucleic acid of  claim 1 . 
     
     
         6 . The modified cell of  claim 5 , wherein said nucleic acid is operably-linked to a promoter functional in said cell. 
     
     
         7 . The modified cell of  claim 6 , wherein said cell is selected from the group consisting of: a fungal cell, a plant cell, an algal cell, and a bacterial cell. 
     
     
         8 . The modified cell of  claim 7 , wherein said cell is a fungal cell selected from the group consisting of: a yeast cell, a mushroom cell, and a filamentous fungus cell. 
     
     
         9 . The modified cell of  claim 8 , wherein said filamentous fungus cell is from a genus selected from the group consisting of:  Chrysosporium, Thielavia, Neurospora, Aureobasidium, Filibasidium, Piromyces, Corynascus, Cryplococcus, Acremonium, Tolypocladium, Scytalidium, Schizophyllum, Sporotrichum, Penicillium, Gibberella, Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola , and  Trichoderma , and anamorphs and teleomorphs thereof. 
     
     
         10 . The modified cell of  claim 9 , wherein said cell comprises at least one additional nucleic acid encoding at least one additional polypeptide, wherein said additional nucleic acid is operably-linked to a promoter functional in said cell. 
     
     
         11 . The modified cell of  claim 10 , wherein said additional polypeptide is an enzyme selected from the group consisting of: cellulase, glucosidase, xylanase, xylosidase, ligninase, glucuronidase, arabinofuranosidase, arabinase, arabinogalactanase, ferulic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, galactosidase, glucoamylase, pectate lyase, chitosanases, exo-β-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase. 
     
     
         12 . A method of preparing a composition comprising one or more polypeptides obtained from the modified cell of  claim 6 , comprising:
 (a) growing said modified cell in a medium;   (b) preparing a cell-free medium; and   (c) concentrating and/or purifying one or more polypeptides from said cell-free medium.   
     
     
         13 . A composition comprising one or more polypeptides isolated from a modified cell of  claim 11 . 
     
     
         14 . The composition of  claim 13 , further comprising at least one additional polypeptide for degrading a lignocellulosic material or a fragment thereof that retains biological activity of said additional polypeptide. 
     
     
         15 . The enzyme composition of  claim 14 , wherein said composition comprises at least one cellobiohydrolase, at least one xylanase, at least one endoglucanase, at least one β-glucosidase, at least one β-xylosidase, and at least one accessory enzyme. 
     
     
         16 . The enzyme composition of  claim 14 , wherein between about 50% and about 70% of the enzymes in the composition are cellobiohydrolases. 
     
     
         17 . The enzyme composition of  claim 14 , wherein between about 10% and about 30% of the enzymes in the composition are xylanases. 
     
     
         18 . The enzyme composition of  claim 14 , wherein between about 5% and about 15% of the enzymes in the composition are endoglucanases. 
     
     
         19 . The enzyme composition of  claim 14 , wherein between about 1% and about 5% of the enzymes in the composition are β-glucosidases. 
     
     
         20 . The enzyme composition of  claim 14 , wherein between about 1% and about 3% of the enzymes in the composition are β-xylosidases. 
     
     
         21 . The enzyme composition of  claim 14 , wherein the composition comprises about 60% cellobiohydrolases, about 20% xylanases, about 10% endoglucanases, about 3% β-glucosidases, about 2% xylosidases, and about 5% accessory enzymes. 
     
     
         22 . The enzyme composition of  claim 21 , wherein the xylanases are selected from the group consisting of: endoxylanases, exoxylanases, and β-xylosidases. 
     
     
         23 . The enzyme composition of  claim 21 , wherein the accessory enzymes include an enzyme selected from the group consisting of: cellulase, glucosidase, xylanase, xylosidase, ligninase, glucuronidase, arabinofuranosidase, arabinase, arabinogalactanase, ferulic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, galactosidase, glucoamylase, pectate lyase, chitosanases, exo-β-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase. 
     
     
         24 . The composition of  claim 13 , wherein the multi-enzyme composition comprises at least one hemicellulase. 
     
     
         25 . The composition of  claim 24 , wherein the hemicellulase is selected from the group consisting of a xylanase, an arabinofuranosidase, an acetyl xylan esterase, a glucuronidase, and endo-galactanase, a mannanase, an endo arabinase, an exo arabinase, an exo-galactanase, a ferulic acid esterase, a galactomannanase, a xylogluconase, and mixtures thereof. 
     
     
         26 . The composition of  claim 25 , wherein the xylanase is selected from the group consisting of endoxylanases, exoxylanase, and β-xylosidase. 
     
     
         27 . The composition of  claim 13 , wherein the multi-enzyme composition comprises at least one cellulase. 
     
     
         28 . The composition of  claim 13 , wherein the composition is a crude fermentation product. 
     
     
         29 . The composition of  claim 13 , wherein the composition is a crude fermentation product that has been subjected to a purification step. 
     
     
         30 . The composition of  claim 13 , further comprising one or more accessory enzymes. 
     
     
         31 . The composition of  claim 30 , wherein the accessory enzymes includes at least one enzyme selected from the group consisting of: cellulase, glucosidase, xylanase, xylosidase, ligninase, glucuronidase, arabinofuranosidase, arabinase, arabinogalactanase, ferulic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, galactosidase, glucoamylase, pectate lyase, chitosanases, exo-β-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase. 
     
     
         32 . The composition of  claim 30 , wherein the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase. 
     
     
         33 . The composition of  claim 30 , wherein the accessory enzyme is a glucoamylase. 
     
     
         34 . The composition of  claim 30 , wherein the accessory enzyme is added as a crude or a semi-purified enzyme mixture. 
     
     
         35 . The composition of  claim 30 , wherein the accessory enzyme is produced by culturing at least one organism on a substrate to produce the enzyme. 
     
     
         36 . An composition comprising at least one polypeptide of  claim 13 , and at least one additional polypeptide for degrading an arabinoxylan-containing material or a fragment thereof that has biological activity. 
     
     
         37 . The composition of  claim 36 , wherein the composition comprises at least one endoxylanase, at least one β-xylosidase, and at least one arabinofuranosidase. 
     
     
         38 . The composition of  claim 36 , wherein the at least one arabinofuranosidase comprises an arabinofuranosidase with specificity towards single substituted xylose residues, an arabinofuranosidase with specificity towards double substituted xylose residues, or a combination thereof. 
     
     
         39 . A purified polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule of  claim 1 . 
     
     
         40 . A purified polypeptide comprising an amino acid sequence encoded by the nucleic acid molecule of  claim 2 .