Novel Fungal Enzymes

• US 20090280105A1
• Filed: 08/02/2007
• Published: 11/12/2009
• Est. Priority Date: 08/02/2007
• Status: Active Grant

• Current Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Verdoes, Jan Comelis, Sinitsyn, Arkady Panteleimonovich, Vlasenko, Elena, Jiang, Zhijie, Gosink, Mark, Gusakov, Alexander Vasilievich, Hinz, Sandra Wihelmina Agnes, Punt, Peter J., Van der Meij, Jacoba
• Granted Patent: US 7,923,236 B2
• Time in Patent Office: Days
• US Class Current: 424/94.610
• CPC Class Codes: A61P 11/06 Antiasthmatics
• 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: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: C11D 3/38645 containing cellulase
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12N 9/2442 Chitinase (3.2.1.14)
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12N 9/2477 Hemicellulases not provided in a preceding group
• CPC Class Codes: C12N 9/248 Xylanases
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 302/01003 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01014 Chitinase (3.2.1.14)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12Y 302/01032 Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
• 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 methods to use the novel enzymes and compositions of such enzymes in a variety of other processes, including washing of clothing, detergent processes, 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;

11, SEQ ID NO;

23, 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;

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, and SEQ ID NO;

94.

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 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 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 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 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 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;

11, SEQ ID NO;

23, 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;

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, and SEQ ID NO;

94.

7. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence consists 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;

10, SEQ ID NO;

12, SEQ ID NO;

22, SEQ ID NO;

24, 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;

41, 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;

90, SEQ ID NO;

91 and SEQ ID NO;

93.

8. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence of (a) encodes a protein comprising the amino acid sequence of SEQ ID NO:

11, wherein the protein has cellobiohydrolase activity.

9. 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;

94 and SEQ ID NO;

23, wherein the protein has endoglucanase activity.

10. 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 and SEQ ID NO;

41, wherein the protein has xylanase activity.

11. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence of (a) encodes a protein comprising an amino acid sequence of SEQ ID NO:

44, wherein the protein has β

-glucosidase activity.

12. 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;

50, SEQ ID NO;

53, 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, and SEQ ID NO;

92, wherein the protein has hemicellulase activity.

13. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence of (a) encodes a protein comprising the amino acid sequence of SEQ ID NO:

50, wherein the protein has glucoamylase activity.

14. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence of (a) encodes a protein comprising the amino acid sequence of SEQ ID NO:

53, wherein the protein has pectate lyase activity.

15. 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;

56 and SEQ ID NO;

59, wherein the protein has chitinase activity.

16. 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;

62, SEQ ID NO;

80, and SEQ ID NO;

83, wherein the protein has acetylxylan esterase activity.

17. 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;

65, SEQ ID NO;

68, and SEQ ID NO;

71, wherein the protein has ferulic acid esterase activity.

18. 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;

74 and SEQ ID NO;

77, wherein the protein has arabinofuranosidase activity.

19. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence of (a) encodes a protein comprising the amino acid sequence of SEQ ID NO:

86, wherein the protein has pectin methyl esterase activity.

20. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence of (a) encodes a protein comprising the amino acid sequence of SEQ ID NO:

89, wherein the protein has endo-arabinase activity.

21. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence of (a) encodes a protein comprising the amino acid sequence of SEQ ID NO:

92, wherein the protein has β

-xylosidase or β

-glucosidase activity.

23. 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.

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

25. An isolated protein comprising an amino acid sequence encoded by the nucleic acid molecule of claim 15.

26. An isolated fusion protein comprising the isolated protein of claim 24 fused to a protein comprising an amino acid sequence that is heterologous to the isolated protein of claim 24.

27. An isolated antibody or antigen binding fragment thereof that selectively binds to the protein of claim 24.

28. A kit for degrading a lignocellulosic material to fermentable sugars comprising at least one isolated protein of claim 24.

29. A detergent comprising at least one isolated protein of claim 24.

30. A composition for the degradation of a lignocellulosic material comprising at least one isolated protein of claim 24.

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

32. The recombinant nucleic acid molecule of claim 31, wherein the recombinant nucleic acid molecule comprises an expression vector.

33. The recombinant nucleic acid molecule of claim 31, wherein the recombinant nucleic acid molecule comprises a targeting vector.

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

35. The isolated host cell of claim 34, wherein the host cell is a fungus.

36. The isolated host cell of claim 34, wherein the host cell is a filamentous fungus.

37. The isolated host cell of claim 36, 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.

38. The isolated host cell of claim 34, wherein the host cell is a bacterium.

41. A method for producing the protein of claim 24, 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.

42. The method of claim 41, further comprising recovering the protein from the cell or from a culture comprising the cell.

43. 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 24.

44. The genetically modified organism of claim 43, wherein the genetically modified organism is a microorganism.

45. The genetically modified organism of claim 44, wherein the microorganism is a filamentous fungus.

46. The genetically modified organism of claim 45, 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.

47. The genetically modified organism of claim 45, 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.

48. The genetically modified organism of claim 43, wherein the organism has been genetically modified to express at least one additional enzyme.

49. The genetically modified organism of claim 48, wherein the additional enzyme is an accessory enzyme selected from the group consisting of:

cellulase, xylanase, ligninase, glucuronidase, arabinofuranosidase, arabinogalactanase, ferulic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, glucoamylase, pectate lyase, chitinase, exo-β

-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase.

50. The genetically modified organism of claim 43, wherein the genetically modified organism is a plant.

51. A recombinant enzyme isolated from the genetically modified microorganism of claim 44.

52. The recombinant enzyme of claim 51, wherein the enzyme has been subjected to a purification step.

53. A crude fermentation product produced by culturing the genetically modified microorganism of claim 44 wherein the crude fermentation product contains the at least one protein of claim 24.

54. A multi-enzyme composition comprising enzymes produced by the genetically modified organism of claim 43, and recovered therefrom.

55. A multi-enzyme composition comprising at least one protein of claim 24, and at least one additional protein for degrading a lignocellulosic material or a fragment thereof that has biological activity.

56. The multi-enzyme composition of claim 55, wherein the at least one additional protein for degrading a lignocellulosic material to fermentable sugars is selected from the group consisting of:

SEQ ID NO;

8, SEQ ID NO;

14, SEQ ID NO;

17, SEQ ID NO;

20, SEQ ID NO;

26 and SEQ ID NO;

47, or a fragment thereof that has biological activity.

57. The multi-enzyme composition of claim 55, 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.

58. The multi-enzyme composition of claim 55, wherein between about 50% and about 70% of the enzymes in the composition are cellobiohydrolases.

59. The multi-enzyme composition of claim 55, wherein between about 10% and about 30% of the enzymes in the composition are xylanases.

60. The multi-enzyme composition of claim 55, wherein between about 5% and about 15% of the enzymes in the composition are endoglucanases.

61. The multi-enzyme composition of claim 55, wherein between about 1% and about 5% of the enzymes in the composition are β

-glucosidases.

62. The multi-enzyme composition of claim 55, wherein between about 1% and about 3% of the enzymes in the composition are β

-xylosidases.

63. The multi-enzyme composition of claim 55, 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.

64. The multi-enzyme composition of claim 57, wherein the xylanases are selected from the group consisting of:

endoxylanases, exoxylanases, and β

-xylosidases.

65. The multi-enzyme composition of claim 57, wherein the accessory enzymes include an enzyme selected from the group consisting of:

ligninase, glucuronidase, arabinofuranosidase, arabinogalactanase, furilic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, glucoamylase, pectate lyase, chitinase, exo-β

-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase.

66. The multi-enzyme composition of claim 55, wherein the composition comprises at least one protein encoded by the nucleic acid molecule of claim 8, at least one protein encoded by the nucleic acid molecule of claim 9, at least one protein encoded by the nucleic acid molecule of claim 10 at least one protein encoded by the nucleic acid molecule of claim 11, and at least one protein encoded by the nucleic acid molecule of claim 12.

67. The multi-enzyme composition of claim 55, wherein the composition comprises:

a) at least one first protein comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of;

SEQ ID NO;

2, SEQ ID NO;

5, SEQ ID NO;

8 and SEQ ID NO;

11 and has cellobiohydrolase activity, or a fragment thereof that has cellobiohydrolase activity;

b) at least one second protein comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of;

SEQ ID NO;

14, SEQ ID NO;

94, SEQ ID NO;

17, SEQ ID NO;

20 and SEQ ID NO;

23 and has endoglucanase activity, or a fragment thereof that has endoglucanase activity; and

c) at least one third protein comprising an amino acid sequence that is at least 95% identical to to an amino acid sequence selected from the group consisting of;

SEQ ID NO;

26, SEQ ID NO;

29, SEQ ID NO;

32, SEQ ID NO;

35, SEQ ID NO;

38 and SEQ ID NO;

41 and has xylanase activity, or a fragment thereof that has xylanase activity.

68. The multi-enzyme composition of claim 67, wherein the composition further comprises a fourth protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

44 and has β

-glucosidase activity, or a fragment thereof that has β

-glucosidase activity.

69. The multi-enzyme composition of claim 68, wherein the composition further comprises at least one fifth protein comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of:

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, and SEQ ID NO;

92, and has hemicellulase or chitinase activity, or a fragment thereof that has hemicellulase or chitinase activity

73. The multi-enzyme composition of claim 55, wherein the multi-enzyme composition comprises at least one hemicellulase.

74. The multi-enzyme composition of claim 73, 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.

75. The multi-enzyme composition of claim 74, wherein the xylanase is selected from the group consisting of endoxylanases, exoxylanase, and β

-xylosidase.

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

77. The multi-enzyme composition of claim 54, wherein the composition is a crude fermentation product.

78. The multi-enzyme composition of claim 54, wherein the composition is a crude fermentation product that has been subjected to a purification step.

79. The multi-enzyme composition of claim 54, further comprising one or more accessory enzymes.

80. The multi-enzyme composition of claim 79, wherein the accessory enzymes includes at least one enzyme selected from the group consisting of:

cellulase, xylanase, ligninase, glucuronidase, arabinofuranosidase, arabinogalactanase, furilic acid esterase, lipase, pectinase, glucomannase, amylase, laminarinase, xyloglucanase, galactanase, glucoamylase, pectate lyase, chitinase, exo-β

-D-glucosaminidase, cellobiose dehydrogenase, and acetylxylan esterase.

81. The multi-enzyme composition of claim 79, wherein the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase.

82. The multi-enzyme composition of claim 79, wherein the accessory enzyme is a glucoamylase.

83. The multi-enzyme composition of claim 79, wherein the accessory enzyme is added as a crude or a semi-purified enzyme mixture.

84. The multi-enzyme composition of claim 79, wherein the accessory enzyme is produced by culturing at least one organism on a substrate to produce the enzyme.

85. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with at least one isolated protein of claim 24.

86. The method of claim 85, wherein the isolated protein is part of a multi-enzyme composition.

87. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with the multi-enzyme composition of claim 55.

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

a) saccharifying a lignocellulosic material with the multi-enzyme composition of claim 55;

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

c) recovering the organic substance from the fermentation.

89. The method of claim 88, wherein the steps of saccharifying and fermenting are performed simultaneously.

90. The method of claim 88, wherein the organic substance is an alcohol, organic acid, ketone, amino acid, or gas.

91. The method of claim 88, wherein the organic substance is an alcohol.

92. The method of claim 91, wherein the alcohol is ethanol.

93. The method of claim 85, wherein the lignocellulosic material is selected from the group consisting of herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper, and pulp and paper mill residue.

94. The method of claim 85, wherein the lignocellulosic material is distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles.

95. The method of claim 85, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles is derived from corn.

96. 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 10% of the fermentable sugars are liberated, wherein the multi-enzyme composition is the multi-enzyme composition of claim 54.

97. The method of claim 96, whereby at least 15% of the sugars are liberated.

98. The method of claim 96, whereby at least 20% of the sugars are liberated.

99. The method of claim 96, whereby at least 23% of the sugars are liberated.

100. The method of claim 96, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles is derived from corn.

101. The method of claim 85, further comprising a pretreatment process for pretreating the lignocellulosic material.

102. The method of claim 101, 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.

103. The method of claim 101, wherein the pretreatment process is selected from the group consisting of organosolv, steam explosion, heat treatment and AFEX.

104. The method of claim 103, wherein the heat treatment comprises heating the lignocellulosic material to 121°

C. for 15 minutes.

105. The method of claim 85, further comprising detoxifying the lignocellulosic material.

106. The method of claim 85, further comprising recovering the fermentable sugar.

107. The method of claim 106, wherein the sugar is selected from the group consisting of glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose and fructose.

108. The method of claim 85, further comprising recovering the contacted lignocellulosic material after the fermentable sugars are degraded.

109. A feed additive comprising the recovered lignocellulosic material of claim 108.

110. The feed additive of claim 109, wherein the protein content of the recovered lignocellulosic material is higher than that of the starting lignocellulosic material.

111. A method of improving the performance of an animal which comprises administering to the animal the feed additive of claim 109.

112. A method for improving the nutritional quality of an animal feed comprising adding the feed additive of claim 109 to an animal feed.

113. A method for stonewashing a fabric, comprising contacting the fabric with at least one isolated protein of claim 24.

114. A method for stonewashing a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 84.

115. The method of claim 113 or claim 114, wherein the fabric is denim.

116. 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 24 or a fragment of at least one isolated protein of claim 24 comprising a cellulose binding domain (CBD) of the protein.

117. 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 54.

118. A method for restoring color to or brightening a fabric, comprising contacting the fabric with at least one isolated protein of claim 24.

119. A method for restoring color to or brightening a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 54.

120. A method of biopolishing, defibrillating, bleaching, dyeing or desizing a fabric, comprising contacting the fabric with at least one isolated protein of claim 24.

121. A method of biopolishing, defibrillating, bleaching, dyeing or desizing a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 54.

122. A method of deinking or biobleaching paper or pulp, comprising contacting the paper or pulp with at least one isolated protein of claim 24.

123. A method of deinking or biobleaching paper or pulp, comprising contacting the paper or pulp with at least one multi-enzyme composition of claim 54.

124. A method for enhancing the cleaning ability of a detergent composition, comprising adding at least one isolated protein of claim 24 to the detergent composition.

125. A method for enhancing the cleaning ability of a detergent composition, comprising adding at least one the multi-enzyme composition claim 54 to the detergent composition.

126. A detergent composition, comprising at least one isolated protein of claim 24 and at least one surfactant.

127. A detergent composition, comprising at least one multi-enzyme composition of claim 54 and at least one surfactant.

128. A method for inhibiting or reducing fungal growth, comprising contacting a fungus or an area susceptible to fungal growth with at least one isolated protein of claim 25.

129. A method for reducing or preventing insect infestation on a plant, comprising contacting the plant with at least one isolated protein of claim 25.

130. A method for reducing or preventing allergic inflammation or asthma, comprising administering at least one isolated protein of claim 25 to a subject.

131. A lysing enzyme for the generation of protoplasts from fingi, comprising at least one isolated protein of claim 25.

132. An insecticide composition, comprising at least one isolated protein of claim 25.

22. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of:

SEQ ID NO;

2 and SEQ ID NO;

5.

39. 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;

10, SEQ ID NO;

12, SEQ ID NO;

22, SEQ ID NO;

24, 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;

41, 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;

90, SEQ ID NO;

91, and SEQ ID NO;

93, or the complement thereof.

40. A kit comprising at least one oligonucleotide of claim 39.

70. A multi-enzyme composition comprising:

a) a first protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

2 and has cellobiohydrolase activity, or a fragment thereof that has cellobiohydrolase activity;

b) a second protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

8 and has cellobiohydrolase activity, or a fragment thereof that has cellobiohydrolase activity;

c) a third protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

94 and has endoglucanase activity, or a fragment thereof that has endoglucanase activity;

d) a fourth protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

29 and has xylanase activity, or a fragment thereof that has xylanase activity; and

e) a fifth protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

44 and has β

-glucosidase activity, or a fragment thereof that has β

-glucosidase activity.

71. The multi-enzyme composition of claim 70, further comprising a sixth protein that has β

-xylosidase activity, or a fragment thereof that has β

-xylosidase activity.

72. The multi-enzyme composition of claim 70, further comprising one or more proteins selected from the group consisting of:

a) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

5 and has cellobiohydrolase activity, or a fragment thereof that has cellobiohydrolase activity;

b) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

20 and has endoglucanase activity, or a fragment thereof that has endoglucanase activity;

c) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

26 and has xylanase activity, or a fragment thereof that has xylanase activity; and

d) a protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO;

47 and has arabinogalactanase activity, or a fragment thereof that has arabinogalactanase activity.