Novel Fungal Enzymes

• US 20090099079A1
• Filed: 09/05/2008
• Published: 04/16/2009
• Est. Priority Date: 09/07/2007
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Van der Meij, Jacoba, Sinitsyn, Arkady Panteleimonovich, Vlasenko, Elena, Jiang, Zhijie, Gosink, Mark, Emalfarb, Mark A., Gusakov, Alexander Vasilievich, Verdoes, Jan Cornelis, Hinz, Sandra Wihelmina Agnes, Punt, Peter J.
• Granted Patent: US 8,551,751 B2
• Time in Patent Office: Days
• US Class Current: 514/1.2
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12Y 301/01072 Acetylxylan esterase (3.1.1.72)
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 302/01037 Xylan 1,4-beta-xylosidase (3.2.1.37)
• CPC Class Codes: C12Y 302/01055 Alpha-N-arabinofuranosidase (3.2.1.55)
• CPC Class Codes: C12Y 302/01139 Alpha-glucuronidase (3.2.1.139)
• CPC Class Codes: C12Y 302/01151 Xyloglucan-specific endo-beta-1,4-glucanase (3.2.1.151), i.e. endoxyloglucanase
• CPC Class Codes: C12Y 302/01155 Xyloglucan-specific exo-beta-1,4-glucanase (3.2.1.155), i.e. exoxyloglucanase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

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.

Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

a) a nucleic acid sequence encoding a protein 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 the protein of (a), wherein the fragment has a biological activity of the protein of (a); and

c) a nucleic acid sequence encoding an amino acid sequence that is at least about 70% identical to an amino acid sequence of (a) and has a biological activity of the protein comprising the amino acid sequence.

2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 90% identical to the amino acid sequence of (a) and has a biological activity of the protein comprising the amino acid sequence.

3. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 95% identical to the amino acid sequence of (a) and has a biological activity of the protein comprising the amino acid sequence.

4. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 97% identical to the amino acid sequence of (a) and has a biological activity of the protein comprising the amino acid sequence.

5. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes an amino acid sequence that is at least about 99% identical to the amino acid sequence of (a) and has a biological activity of the protein comprising the amino acid sequence.

6. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes a protein 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.

7. 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.

8. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence of (a) encodes a protein comprising an amino acid sequence selected from the group consisting of:

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;

95, SEQ ID NO;

98, SEQ ID NO;

101, SEQ ID NO;

104, and SEQ ID NO;

107, wherein the protein has cellulolytic enhancing activity.

9. An isolated nucleic acid molecule comprising a nucleic acid sequence that is fully complementary to the nucleic acid sequence of the nucleic acid molecule of claim 1.

10. An isolated protein comprising an amino acid sequence encoded by the nucleic acid molecule of claim 1.

11. An isolated protein comprising an amino acid sequence encoded by the nucleic acid molecule of claim 8.

12. An isolated fusion protein comprising the isolated protein of claim 10 fused to a protein comprising an amino acid sequence that is heterologous to the isolated protein of claim 10.

13. An isolated antibody or antigen binding fragment thereof that selectively binds to the protein of claim 10.

14. A kit for degrading a lignocellulosic material to fermentable sugars comprising at least one isolated protein of claim 10.

15. A detergent comprising at least one isolated protein of claim 10.

16. A composition for the degradation of a lignocellulosic material comprising at least one isolated protein of claim 10.

17. A recombinant nucleic acid molecule comprising the isolated nucleic acid molecule of claim 1, operatively linked to at least one expression control sequence.

18. The recombinant nucleic acid molecule of claim 17, wherein the recombinant nucleic acid molecule comprises an expression vector.

19. The recombinant nucleic acid molecule of claim 17, wherein the recombinant nucleic acid molecule comprises a targeting vector.

20. An isolated host cell transfected with the nucleic acid molecule of claim 1.

21. The isolated host cell of claim 20, wherein the host cell is selected from the group consisting of:

a fungal cell, a plant cell, an algal cell, and a bacterium.

22. The isolated host cell of claim 20, wherein the host cell is selected from the group consisting of:

yeast, mushroom, or a filamentous fungus.

23. The isolated host cell of claim 22, wherein the filamentous fungus 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.

24. The isolated host cell of claim 20, wherein the host cell is a bacterium.

27. A method for producing the protein of claim 10, comprising culturing a cell that has been transfected with a nucleic acid molecule comprising a nucleic acid sequence encoding the protein, and expressing the protein with the transfected cell.

28. The method of claim 27, further comprising recovering the protein from the cell or from a culture comprising the cell.

29. A genetically modified organism comprising components suitable for degrading a lignocellulosic material to fermentable sugars, wherein the organism has been genetically modified to express at least one protein of claim 10.

30. The genetically modified organism of claim 29, wherein the genetically modified organism is selected from the group consisting of:

plants, algae, fungi, and bacteria.

31. The genetically modified organism of claim 30, wherein the fungus is selected from the group consisting of:

yeast, mushroom and filamentous fungus.

32. The genetically modified organism of claim 31, wherein the filamentous fungus is from a genus selected from the group consisting of:

Chrysosporium, Thielavia, Neurospora, Aureobasidium, Filibasidium, Piromyces, Corynascus, Cryplococcus, Acremonium, Tolypocladium, Scytalidium, Schizophyllum, Sporotrichum, Penicillium, Talaromyces, Gibberella, Myceliophthora, Mucor, Aspergillus, Fusarium, Humicola, and Trichoderma.

33. The genetically modified organism of claim 31, wherein the filamentous fungus is selected from the group consisting of:

Trichoderma reesei, Chrysosporium lucknowense, Aspergillus japonicus, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, and Talaromyces flavus.

34. The genetically modified organism of claim 29, wherein the organism has been genetically modified to express at least one additional enzyme.

35. The genetically modified organism of claim 34, wherein the additional enzyme is an accessory 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.

36. The genetically modified organism of claim 29, wherein the genetically modified organism is a plant.

37. A recombinant enzyme isolated from the genetically modified microorganism of claim 29.

38. The recombinant enzyme of claim 37, wherein the enzyme has been subjected to a purification step.

39. A crude fermentation product produced by culturing the cells from the genetically modified organism of claim 29, wherein the crude fermentation product contains the at least one protein of claim 10.

40. A multi-enzyme composition comprising enzymes produced by the genetically modified organism of claim 29, and recovered therefrom.

41. A multi-enzyme composition comprising at least one protein of claim 10, and at least one additional protein for degrading a lignocellulosic material or a fragment thereof that has biological activity.

42. The multi-enzyme composition of claim 41, wherein the 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.

43. The multi-enzyme composition of claim 41, wherein between about 50% and about 70% of the enzymes in the composition are cellobiohydrolases.

44. The multi-enzyme composition of claim 41, wherein between about 10% and about 30% of the enzymes in the composition are xylanases.

45. The multi-enzyme composition of claim 41, wherein between about 5% and about 15% of the enzymes in the composition are endoglucanases.

46. The multi-enzyme composition of claim 41, wherein between about 1% and about 5% of the enzymes in the composition are β

-glucosidases.

47. The multi-enzyme composition of claim 41, wherein between about 1% and about 3% of the enzymes in the composition are β

-xylosidases.

48. The multi-enzyme composition of claim 41, wherein the composition comprises about 60% cellobiohydrolases, about 20% xylanases, about 10% endoglucanases, about 3% β

-glucosidases, about 2% β

-xylosidases, and about 5% accessory enzymes.

49. The multi-enzyme composition of claim 42, wherein the xylanases are selected from the group consisting of:

endoxylanases, exoxylanases, and β

-xylosidases.

50. The multi-enzyme composition of claim 42, 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.

51. The multi-enzyme composition of claim 40, wherein the multi-enzyme composition comprises at least one hemicellulase.

52. The multi-enzyme composition of claim 51, 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.

53. The multi-enzyme composition of claim 52, wherein the xylanase is selected from the group consisting of endoxylanases, exoxylanase, and β

-xylosidase.

54. The multi-enzyme composition of claim 40, wherein the multi-enzyme composition comprises at least one cellulase.

55. The multi-enzyme composition of claim 40, wherein the composition is a crude fermentation product.

56. The multi-enzyme composition of claim 40, wherein the composition is a crude fermentation product that has been subjected to a purification step.

57. The multi-enzyme composition of claim 40, further comprising one or more accessory enzymes.

58. The multi-enzyme composition of claim 57, 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.

59. The multi-enzyme composition of claim 57, wherein the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase.

60. The multi-enzyme composition of claim 57, wherein the accessory enzyme is a glucoamylase.

61. The multi-enzyme composition of claim 57, wherein the accessory enzyme is added as a crude or a semi-purified enzyme mixture.

62. The multi-enzyme composition of claim 57, wherein the accessory enzyme is produced by culturing at least one organism on a substrate to produce the enzyme.

63. A multi-enzyme composition comprising at least one protein of claim 10, and at least one additional protein for degrading an arabinoxylan-containing material or a fragment thereof that has biological activity.

64. The multi-enzyme composition of claim 63, wherein the composition comprises at least one endoxylanase, at least one β

-xylosidase, and at least one arabinofuranosidase.

65. The multi-enzyme composition of claim 64, 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.

66. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with at least one isolated protein of claim 10.

67. The method of claim 66, further comprising contacting the lignocellulosic material with at least one additional isolated protein comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of:

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;

95, SEQ ID NO;

98, SEQ ID NO;

101, SEQ ID NO;

104, and SEQ ID NO;

107, wherein the at least one additional protein has cellulolytic enhancing activity.

68. The method of claim 66, wherein the isolated protein is part of a multi-enzyme composition.

69. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with at least one multi-enzyme composition of claim 40.

70. A method for producing an organic substance, comprising:

a) saccharifying a lignocellulosic material with a multi-enzyme composition of claim 40;

b) fermenting the saccharified lignocellulosic material obtained with one or more fermentating microorganisms; and

c) recovering the organic substance from the fermentation.

71. The method of claim 70, wherein the steps of saccharifying and fermenting are performed simultaneously.

72. The method of claim 70, wherein the organic substance is an alcohol, organic acid, ketone, amino acid, or gas.

73. The method of claim 70, wherein the organic substance is an alcohol.

74. The method of claim 73, wherein the alcohol is ethanol.

75. The method of claim 66, wherein the lignocellulosic material is selected from the group consisting of consisting of herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper, and pulp and paper mill residue.

76. The method of claim 66, wherein the lignocellulosic material is distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles.

77. The method of claim 66, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles is derived from corn.

78. A method for degrading a lignocellulosic material consisting of distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles to sugars, the method comprising contacting the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles with a multi-enzyme composition, whereby at least about 10% of the fermentable sugars are liberated, wherein the multi-enzyme composition is the multi-enzyme composition of claim 40.

79. The method of claim 78, whereby at least about 15% of the sugars are liberated.

80. The method of claim 78, whereby at least about 20% of the sugars are liberated.

81. The method of claim 78, whereby at least about 23% of the sugars are liberated.

82. The method of claim 78, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles is derived from corn.

83. The method of claim 66, further comprising a pretreatment process for pretreating the lignocellulosic material.

84. The method of claim 83, wherein the pretreatment process is selected from the group consisting of physical treatment, metal ion, ultraviolet light, ozone, organosolv treatment, steam explosion treatment, lime impregnation with steam explosion treatment, hydrogen peroxide treatment, hydrogen peroxide/ozone (peroxone) treatment, acid treatment, dilute acid treatment, and base treatment.

85. The method of claim 83, wherein the pretreatment process is selected from the group consisting of organosolv, steam explosion, heat treatment and AFEX.

86. The method of claim 85, wherein the heat treatment comprises heating the lignocellulosic material to 121°

C. for 15 minutes.

87. The method of claim 66, further comprising detoxifying the lignocellulosic material.

88. The method of claim 66, further comprising recovering the fermentable sugar.

89. The method of claim 88, wherein the sugar is selected from the group consisting of glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose and fructose.

90. The method of claim 66, further comprising recovering the contacted lignocellulosic material after the fermentable sugars are degraded.

91. A feed additive comprising the recovered lignocellulosic material of claim 90.

92. The feed additive of claim 91, wherein the protein content of the recovered lignocellulosic material is higher than that of the starting lignocellulosic material.

93. A method of improving the performance of an animal which comprises administering to the animal the feed additive of claim 91.

94. A method for improving the nutritional quality of an animal feed comprising adding the feed additive of claim 91 to an animal feed.

95. A method for stonewashing a fabric, comprising contacting the fabric with at least one isolated protein of claim 10.

96. A method for stonewashing a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 40.

97. The method of claim 95, wherein the fabric is denim.

98. A method for enhancing the softness or feel of a fabric or depilling a fabric, comprising contacting the fabric with at least one isolated protein of claim 10 or a fragment of at least one isolated protein of claim 10 comprising a cellulose binding module (CBM) of the protein.

99. A method for enhancing the softness or feel of a fabric or depilling a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 40.

100. A method for restoring color to or brightening a fabric, comprising contacting the fabric with at least one isolated protein of claim 10.

101. A method for restoring color to or brightening a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 40.

102. A method of biopolishing, defibrillating, bleaching, dyeing or desizing a fabric, comprising contacting the fabric with at least one isolated protein of claim 10.

103. A method of biopolishing, defibrillating, bleaching, dyeing or desizing a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 40.

104. A method of biorefining, deinking or biobleaching paper or pulp, comprising contacting the paper or pulp with at least one isolated protein of claim 10.

105. A method of biorefining, deinking or biobleaching paper or pulp, comprising contacting the paper or pulp with at least one multi-enzyme composition of claim 40.

106. A method for enhancing the cleaning ability of a detergent composition, comprising adding at least one isolated protein of claim 10 to the detergent composition.

107. A method for enhancing the cleaning ability of a detergent composition, comprising adding at least one multi-enzyme composition of claim 40 to the detergent composition.

108. A detergent composition, comprising at least one isolated protein of claim 10 and at least one surfactant.

109. A detergent composition, comprising at least one multi-enzyme composition of claim 40 and at least one surfactant.

115. A method for releasing cellular contents comprising contacting a cell with at least one isolated protein of claim 10.

116. The method of claim 115, wherein the cell is selected from the group consisting of:

a bacterium, an algal cell, a fungal cell or a plant cell.

117. The method of claim 115, where the cell is an algal cell.

118. The method of claim 115, wherein contacting the cell with at least one isolated protein of claim 10 degrades the cell wall.

119. The method of claim 115, wherein the cellular contents are selected from the group consisting of:

alcohols and oils.

120. A composition for degrading cell walls comprising at least one isolated protein of claim 10.

121. A method for improving the nutritional quality of food comprising adding to the food at least one isolated protein of claim 10.

122. A method for improving the nutritional quality of food comprising pretreating the food with at least one isolated protein of claim 10.

123. A method for improving the nutritional quality of animal feed comprising adding to the animal feed at least one isolated protein of claim 10.

124. A method for improving the nutritional quality of animal feed comprising pretreating the feed with at least one isolated protein of claim 10.

125. A genetically modified organism comprising at least one nucleic acid molecule encoding at least one protein of claim 10, in which the activity of one or more of the proteins of claim 10 is upregulated, the activity of one or more of the proteins of claim 10 is downregulated, or the activity of one or more of the proteins of claim 10 is upregulated and the activity of one or more of the proteins of claim 10 is downregulated.

25. An oligonucleotide consisting essentially of at least 12 consecutive nucleotides of 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 or the complement thereof.

26. A kit comprising at least one oligonucleotide of claim 25.

110. A method for inhibiting or reducing fungal growth, comprising contacting a fungus or an area susceptible to fungal growth with an isolated protein comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

188.

111. A method for reducing or preventing insect infestation on a plant, comprising contacting the plant with an isolated protein comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

188.

112. A method for reducing or preventing allergic inflammation or asthma, comprising administering an isolated protein comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

188.

113. A lysing enzyme for the generation of protoplasts from fungi, comprising an isolated protein comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

188.

114. An insecticide composition, comprising an isolated protein comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:

188.