Fungal enzymes

• US 7,923,236 B2
• Filed: 08/02/2007
• Issued: 04/12/2011
• Est. Priority Date: 08/02/2007
• Status: Expired due to Fees

• Current Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Jiang, Zhijie, Gosink, Mark, Gusakov, Alexander Vasilievich, Verdoes, Jan Cornelis, Hinz, Sandra Wihelmina Agnes, Punt, Peter J., Sinitsyn, Arkady Panteleimonovich, Vlasenko, Elena
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US11/833,133
• Publication Number:
  US 20090280105A1
• Time in Patent Office: 1,349 Days
• Field of Search: 435/209, 435/252.3, 435/254.11, 435/254.5, 435/254.6, 435/320.1, 435/69.1, 435/72, 435/128, 435/148, 435/161, 435/163, 536/23.2, 530/350, 510/392, 162/174, 162/72, 810/7, 815/9, 863/6
• US Class Current: 435/209
• 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 enzymes and 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 protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

44 and having β

-glucosidase activity or a catalytic domain of SEQ ID NO;

44, wherein the catalytic domain comprises from about amino acid 87 to about amino acid 643 of SEQ ID NO;

44 and having β

-glucosidase activity.

2. An isolated fusion protein comprising the isolated protein of claim 1 fused to a protein comprising an amino acid sequence that is heterologous to the isolated protein of claim 1.

3. A kit for degrading a lignocellulosic material to fermentable sugars comprising at least one isolated protein of claim 1.

4. A detergent comprising at least one isolated protein of claim 1.

5. A composition for the degradation of a lignocellulosic material comprising at least one isolated protein of claim 1.

6. A recombinant enzyme isolated from a genetically modified microorganism, wherein the recombinant enzyme comprises an amino acid sequence of claim 1.

7. The recombinant enzyme of claim 6, wherein the enzyme has been subjected to a purification step.

8. A crude fermentation product produced by culturing a genetically modified microorganism, wherein the crude fermentation product contains at least one protein of claim 1.

9. A multi-enzyme composition comprising at least one protein of claim 1, and at least one additional protein for degrading a lignocellulosic material or a fragment thereof that has biological activity.

10. The multi-enzyme composition of claim 9, 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.

11. The multi-enzyme composition of claim 9, wherein between about 50% and about 70% of the enzymes in the composition are cellobiohydrolases.

12. The multi-enzyme composition of claim 9, wherein between about 10% and about 30% of the enzymes in the composition are xylanases.

13. The multi-enzyme composition of claim 9, wherein between about 5% and about 15% of the enzymes in the composition are endoglucanases.

14. The multi-enzyme composition of claim 9, wherein between about 1% and about 5% of the enzymes in the composition are β

-glucosidases.

15. The multi-enzyme composition of claim 9, wherein between about 1% and about 3% of the enzymes in the composition are β

-xylosidases.

16. The multi-enzyme composition of claim 9, 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.

17. The multi-enzyme composition of claim 10, wherein the xylanases are selected from the group consisting of:

endoxylanases, exoxylanases, and β

-xylosidases.

18. The multi-enzyme composition of claim 10, 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.

21. The multi-enzyme composition of claim 9, wherein the multi-enzyme composition comprises at least one hemicellulase.

22. The multi-enzyme composition of claim 21, 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.

23. The multi-enzyme composition of claim 22, wherein the xylanase is selected from the group consisting of endoxylanases, exoxylanase, and β

-xylosidase.

24. The multi-enzyme composition of claim 9, wherein the multi-enzyme composition comprises at least one cellulase.

25. The multi-enzyme composition of claim 9, wherein the composition is a crude fermentation product.

26. The multi-enzyme composition of claim 9, wherein the composition is a crude fermentation product that has been subjected to a purification step.

27. The multi-enzyme composition of claim 9, further comprising one or more accessory enzymes.

28. The multi-enzyme composition of claim 27, 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.

29. The multi-enzyme composition of claim 27, wherein the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase.

30. The multi-enzyme composition of claim 27, wherein the accessory enzyme is a glucoamylase.

31. The multi-enzyme composition of claim 27, wherein the accessory enzyme is added as a crude or a semi-purified enzyme mixture.

32. The multi-enzyme composition of claim 27, wherein the accessory enzyme is produced by culturing an organism on a substrate to produce the enzyme.

33. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with the multi-enzyme composition of claim 9.

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

a) saccharifying a lignocellulosic material with the multi-enzyme composition of claim 9;

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

c) recovering the organic substance from the fermentation.

35. The method of claim 34, wherein the steps of saccharifying and fermenting are performed simultaneously.

36. The method of claim 34, wherein the organic substance is an alcohol, organic acid, ketone, amino acid, or gas.

37. The method of claim 34, wherein the organic substance is an alcohol.

38. The method of claim 37, wherein the alcohol is ethanol.

39. 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 9.

40. The method of claim 39, whereby at least 15% of the sugars are liberated.

41. The method of claim 39, whereby at least 20% of the sugars are liberated.

42. The method of claim 39, whereby at least 23% of the sugars are liberated.

43. The method of claim 39, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles is derived from corn.

44. A method for stonewashing a fabric, comprising contacting the fabric with at least one isolated protein of claim 1.

45. A method for stonewashing a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 9.

46. The method of claim 44 or claim 45, wherein the fabric is denim.

47. 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 1.

48. 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 9.

49. A method for restoring color to or brightening a fabric, comprising contacting the fabric with at least one isolated protein of claim 1.

50. A method for restoring color to or brightening a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 9.

51. A method of biopolishing, defibrillating, bleaching, dyeing or desizing a fabric, comprising contacting the fabric with at least one isolated protein of claim 1.

52. A method of biopolishing, defibrillating, bleaching, dyeing or desizing a fabric, comprising contacting the fabric with at least one multi-enzyme composition of claim 9.

53. A method of treating, biorefining, deinking or biobleaching paper or pulp, comprising contacting the paper or pulp with at least one isolated protein of claim 1.

54. A method of treating, biorefining, deinking or biobleaching paper or pulp, comprising contacting the paper or pulp with at least one multi-enzyme composition of claim 9.

55. A method for enhancing the cleaning ability of a detergent composition, comprising adding at least one isolated protein of claim 1 to the detergent composition.

56. A method for enhancing the cleaning ability of a detergent composition, comprising adding the multi-enzyme composition claim 9 to the detergent composition.

57. A detergent composition, comprising at least one isolated protein of claim 1 and at least one surfactant.

58. A detergent composition, comprising at least one multi-enzyme composition of claim 9 and at least one surfactant.

19. A multi-enzyme composition comprising:

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

44 and having β

-glucosidase activity or a catalytic domain of SEQ ID NO;

44, wherein the catalytic domain comprises from about amino acid 87 to about amino acid 643 of SEQ ID NO;

44 and having β

-glucosidase activity;

b) at least one protein, or a fragment thereof that has cellobiohydrolase activity;

c) at least one protein, or a fragment thereof that has endoglucanase activity; and

d) at least one protein, or a fragment thereof that has xylanase activity.

20. The multi-enzyme composition of claim 19, further comprising at least one protein that has β

-xylosidase activity, or a fragment thereof that has β

-xylosidase activity.