ENZYME COMPOSITIONS FOR THE IMPROVED ENZYMATIC HYDROLYSIS OF CELLULOSE AND METHODS OF USING SAME

• US 20090209009A1
• Filed: 06/22/2007
• Published: 08/20/2009
• Est. Priority Date: 06/22/2006
• Status: Active Grant

• Current Assignee: Iogen Energy Corporation (Iogen Corporation)
• Original Assignee: Iogen Energy Corporation (Iogen Corporation)
• Inventors: Tolan, Jeffrey, White, Theresa, Tomashek, John
• Granted Patent: US 8,318,461 B2
• Time in Patent Office: Days
• US Class Current: 435/105
• 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 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

A process for the enzymatic hydrolysis of cellulose to produce a hydrolysis product comprising glucose from a pretreated lignocellulosic feedstock and enzymes for use in the process are provided. The process comprises hydrolyzing an aqueous slurry of a pretreated lignocellulosic feedstock with cellulase enzymes, one or more than one β-glucosidase enzyme and a binding agent for binding the β-glucosidase enzyme to fiber solids present in the aqueous slurry. During the hydrolysis, both the cellulase enzyme and β-glucosidase enzyme bind to the fiber solids. The hydrolysis is performed in a solids-retaining hydrolysis reactor so that unhydrolyzed fiber solids and bound enzyme are retained in the reactor longer than the aqueous phase of the slurry.

Claims

1. An enzyme composition for the enzymatic hydrolysis of cellulose to produce a hydrolysis product comprising glucose from a pretreated lignocellulosic feedstock, the enzyme composition comprising cellulase enzymes, one or more than one β

-glucosidase enzyme and a binding agent for binding the β

-glucosidase enzyme to the pretreated lignocellulosic feedstock, wherein the hydrolysis is carried out by;

(i) providing an aqueous slurry of the pretreated lignocellulosic feedstock, said aqueous slurry comprising fiber solids and an aqueous phase;

(ii) hydrolyzing the aqueous slurry with the enzyme composition comprising cellulase enzymes, one or more than one β

-glucosidase enzyme and the binding agent in a solids-retaining hydrolysis reactor to produce said hydrolysis product comprising glucose, wherein the cellulase enzymes and the one or more than one β

-glucosidase enzyme bind to the fiber solids; and

(iii) withdrawing unhydrolyzed fiber solids and the aqueous phase, which comprises hydrolysis product, from the solids-retaining hydrolysis reactor,

wherein the unhydrolyzed fiber solids are retained in the solids-retaining hydrolysis reactor for a mean time of about 6 hours to about 148 hours longer than the aqueous phase.

2. The enzyme composition according to claim 1, wherein the binding agent is a carbohydrate-binding module operably linked to the one or more than one β

-glucosidase enzyme.

3. The enzyme composition according to claim 2, wherein the carbohydrate-binding module is a cellulose-binding domain.

4. The enzyme composition according to claim 1, wherein the cellulase enzymes are produced by Aspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.

5. The enzyme composition according to claim 4, wherein the cellulase enzymes are produced by Trichoderma.

6. The enzyme composition according to claim 1, wherein the one or more than one glucosidase enzyme is produced by Aspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.

7. The enzyme composition according to claim 6, wherein the one or more than one glucosidase enzyme is produced by Trichoderma or Aspergillus.

8. The enzyme composition according to claim 3, wherein the cellulose-binding domain is a Family I cellulose-binding domain.

9. The enzyme composition according to claim 3, wherein the cellulose-binding domain is a bacterial or fungal cellulose-binding domain.

10. The enzyme composition according to claim 3, wherein the one or more than one glucosidase enzyme comprises a linker which operably links the cellulose-binding domain to the β

-glucosidase enzyme.

11. The enzyme composition according to claim 1, wherein the one or more than one β

-glucosidase enzyme is naturally occurring.

12. The enzyme composition according to claim 1, wherein the one or more than one β

-glucosidase enzyme is a genetically modified fusion protein.

13. The enzyme composition according to claim 3, wherein about 75% to about 100% (w/w) of the total β

-glucosidase enzyme present in the enzyme composition comprises a cellulose-binding domain.

14. The enzyme composition according to claim 13, wherein about 90% to about 100% (w/w) of the total β

-glucosidase enzyme present in the enzyme composition comprises a cellulose-binding domain.

15. The enzyme composition according to claim 1, wherein the cellulase enzymes comprise a cellobiohydrolase enzyme selected from the group consisting of CBHI and CBHII cellulase enzymes, and combinations thereof, and an endoglucanase enzyme selected from the group consisting of EGI, EGII, EGIII, EGIV, EGV and EGVI cellulase enzymes, and combinations thereof.

16. The enzyme composition according to claim 1, wherein about 75% to about 100% (w/w) of the total cellulase enzymes present in the enzyme composition bind to the fiber solids in the step of hydrolyzing (step (ii)).

17. The enzyme composition according to claim 1, further comprising a step of separating the unhydrolyzed solids from the hydrolysis product comprising glucose to produce separated solids and an aqueous solution comprising glucose.

18. The enzyme composition according to claim 17, further comprising re-suspending the separated solids in an aqueous solution, and continuing the hydrolysis.

19. The use of an enzyme composition for the enzymatic hydrolysis of cellulose to produce a hydrolysis product comprising glucose from a pretreated lignocellulosic feedstock, the enzyme composition comprising cellulase enzymes, one or more than one β

-glucosidase enzyme and a binding agent for binding the β

-glucosidase enzyme to the pretreated lignocellulosic feedstock, wherein the use of the enzyme composition comprises;

(i) providing an aqueous slurry of the pretreated lignocellulosic feedstock, said aqueous slurry comprising fiber solids and an aqueous phase;

(ii) hydrolyzing the aqueous slurry with the enzyme composition comprising cellulase enzymes, one or more than one β

-glucosidase enzyme and the binding agent in a solids-retaining hydrolysis reactor to produce said hydrolysis product comprising glucose, wherein the cellulase enzymes and the one or more than one β

-glucosidase enzyme bind to the fiber solids; and

(iii) withdrawing unhydrolyzed fiber solids and the aqueous phase, which comprises hydrolysis product, from the solids-retaining hydrolysis reactor,

wherein the unhydrolyzed fiber solids are retained in the solids-retaining hydrolysis reactor for a meant time of about 6 hours to about 148 hours longer than the aqueous phase.

20. The use of the enzyme composition according to claim 19, wherein, in the step of hydrolyzing (step (ii)), the binding agent is a carbohydrate-binding module operably linked to the one or more than one β

-glucosidase enzyme.

21. The use of the enzyme composition according to claim 20, wherein the carbohydrate-binding module is a cellulose-binding domain.

22. The use of the enzyme composition according to claim 19, wherein the solids-retaining hydrolysis reactor is part of a hydrolysis system comprising two or more hydrolysis reactors and the hydrolysis reactors are operated in series, in parallel, or a combination thereof.

23. The use of the enzyme composition according to claim 19, wherein the solids-retaining hydrolysis reactor is a settling reactor and wherein, during the step of hydrolyzing (step (ii)), at least a portion of the fiber solids settle in the settling reactor.

24. The enzyme composition according to claim 23, wherein the settling reactor is a tower and wherein, in the step of hydrolyzing (step (ii)), the aqueous slurry flows upward through the tower.

25. The use of the enzyme composition according to claim 19, wherein, in the step of hydrolyzing (step (ii)), the cellulase enzymes are produced by Aspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.

26. The use of the enzyme composition according to claim 25, wherein the cellulase enzymes are produced by Trichoderma.

27. The use of the enzyme composition according to claim 19, wherein, in the step of hydrolyzing (step (ii)), the one or more than one β

-glucosidase enzyme is produced by Aspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.

28. The use of the enzyme composition according to claim 27, wherein the one or more than one β

-glucosidase enzyme is produced by Trichoderma or Aspergillus.

29. The use of the enzyme composition according to claim 21, wherein, in the step of hydrolyzing (step (ii)), the cellulose-binding domain is a Family I cellulose-binding domain.

30. The use of the enzyme composition according to claim 21, wherein, in the step of hydrolyzing (step (ii)), the cellulose-binding domain is a bacterial or fungal cellulose-binding domain.

31. The use of the enzyme composition according to claim 21, wherein, in the step of hydrolyzing (step (ii)), the one or more than one β

-glucosidase enzyme comprises a linker which operably links the cellulose-binding domain to the β

-glucosidase enzyme.

32. The use of the enzyme composition according to claim 19, wherein, in the step of hydrolyzing (step (ii)), the one or more than one β

-glucosidase enzyme is naturally occurring.

33. The use of the enzyme composition according to claim 19, wherein, in the step of hydrolyzing (step (ii)), the one or more than one β

-glucosidase enzyme is a genetically modified fusion protein.

34. The use of the enzyme composition according to claim 19, wherein, in the step of hydrolyzing (step (ii)), about 75% to about 100% (w/w) of the total β

-glucosidase enzyme present in the enzyme composition comprises a cellulose-binding domain.

35. The use of the enzyme composition according to claim 34, wherein, in the step of hydrolyzing (step (ii)), about 90% to about 100% (w/w) of the total β

-glucosidase enzyme present in the enzyme composition comprises a cellulose-binding domain.

36. The use of the enzyme composition according to claim 19, wherein, in the step of hydrolyzing (step (ii)), the cellulase enzymes comprise a cellobiohydrolase selected from the group consisting of CBHI and CBHII cellulase enzymes, and combinations thereof, and an endoglucanase selected from the group consisting of EGI, EGII, EGIII, EGIV, EGV and EGVI cellulase enzymes, and combinations thereof.

37. The use of the enzyme composition according to claim 19, wherein, in the step of hydrolyzing (step (ii)), about 75% to about 100% (w/w) of the total cellulase enzymes present in the enzyme composition bind to the fiber solids in step (ii).

38. The use of the enzyme composition according to claim 19, further comprising a step of separating the unhydrolyzed solids from the hydrolysis product comprising glucose to obtain separated solids and an aqueous solution comprising glucose.

39. The use of the enzyme composition according to claim 38, further comprising re-suspending the separated solids in an aqueous solution, and continuing the hydrolysis.

52. The process according to claim 39, wherein the process is a batch process.

40. A process for the enzymatic hydrolysis of cellulose to produce a hydrolysis product comprising glucose from a pretreated lignocellulosic feedstock, the process comprising:

(i) providing an aqueous slurry of the pretreated lignocellulosic feedstock, said aqueous slurry comprising fiber solids and an aqueous phase;

(ii) hydrolyzing the aqueous slurry with cellulase enzymes, one or more than one β

-glucosidase enzyme and a binding agent for binding the β

-glucosidase enzyme to the fiber solids in a solids-retaining hydrolysis reactor to produce said hydrolysis product comprising glucose, wherein the cellulase enzymes and the one or more than one β

-glucosidase enzyme bind to the fiber solids; and

(iii) withdrawing unhydrolyzed solids and the aqueous phase, which comprises hydrolysis product, from the solids-retaining hydrolysis reactor,

wherein the unhydrolyzed fiber solids are retained in the solids-retaining hydrolysis reactor for a meant time of about 6 hours to about 148 hours longer than the aqueous phase.

41. The process according to claim 40, wherein, in the step of hydrolyzing (step (ii)), the binding agent is a carbohydrate-binding module operably linked to the one or more than one β

-glucosidase enzyme.

42. The process according to claim 41, wherein the carbohydrate-binding module is a cellulose-binding domain.

43. The process according to claim 40, wherein, in the step of providing (step (i)), the aqueous slurry has a suspended or undissolved fiber solids content of about 3% to about 30% (w/w).

44. The process according to claim 40, further comprising a step of separating the hydrolyzed solids from the hydrolysis product comprising glucose to produce separated solids and an aqueous solution comprising glucose.

45. The process according to claim 40, wherein the solids-retaining hydrolysis reactor is part of a hydrolysis system comprising two or more hydrolysis reactors, and the hydrolysis reactors are operated in series, in parallel, or a combination thereof.

46. The process according to claim 44, wherein the unhydrolyzed solids are separated from the hydrolysis product by microfiltration, centrifugation, vacuum filtration or pressure filtration.

47. The process according to claim 46, wherein the unhydrolyzed solids are separated from the hydrolysis product by microfiltration.

48. The process according to claim 40, wherein the aqueous slurry is concentrated prior to the step of hydrolyzing (step (ii)).

49. The process according to claim 45, wherein the hydrolysis system comprises one or more than one hydrolysis reactor selected from the group consisting of an agitated tank, an unmixed tank, an agitated tower and an unmixed tower.

50. The process according to claim 49, wherein the agitated tower is an upflow tower.

51. The process according to claim 49, wherein the unmixed tower is an upflow tower.

53. The process according to claim 40, wherein the process is a continuous process with continuous feeding of the aqueous slurry and continuous withdrawal of the unhydrolyzed solids and the aqueous phase comprising the hydrolysis product.

54. The process according to claim 40, wherein, in the step of hydrolyzing (step (ii)), about 70% to about 100% of cellulose in the fiber solids is converted to glucose.

55. The process according to claim 40, further comprising a step of separating the unhydrolyzed solids from the hydrolysis product comprising glucose to produce separated solids and a first aqueous solution comprising glucose, re-suspending the separated solids in an aqueous solution to produce a re-suspended slurry, continuing the hydrolysis of the re-suspended slurry in a second hydrolysis and obtaining a second aqueous solution comprising glucose from said second hydrolysis.

56. The process according to claim 55, wherein the re-suspended slurry has a solids concentration of about 10% to about 15% (w/w).

57. The process according to claim 40, wherein, in the step of providing (step (i)), the pretreated lignocellulosic feedstock is obtained from wheat straw, oat straw, barley straw, corn stover, soybean stover, canola straw, rice straw, sugar cane, bagasse, switch grass, reed canary grass, cord grass, or miscanthus.

58. The process according to claim 40, wherein, in the step of hydrolyzing (step (ii)), the cellulase enzymes are added at a dosage of about 1.0 to about 40.0 IU per gram of cellulose.

59. The process according to claim 40, wherein, in the step of hydrolyzing (step (ii)), the one or more than one β

-glucosidase enzyme is added at a dosage of 35 to about 200 IU per gram of cellulose.

60. The process according to claim 40, wherein, in the step of hydrolyzing (step (ii)), the cellulase enzymes are produced by Aspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.

61. The process according to claim 40, wherein, in the step of hydrolyzing (step (ii)), the β

-glucosidase enzyme is produced by Aspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or a combination thereof.

62. The process according to claim 61, wherein the β

-glucosidase enzyme is produced by Aspergillus or Trichoderma.

63. The process according to claim 40, wherein the step of hydrolyzing (step (ii)) is carried out for a total residence time of the aqueous phase of about 12 to about 200 hours.

64. The process according to claim 55, wherein the step of continuing the hydrolysis is carried out for about 12 to about 200 hours.

65. The process according to claim 55, further comprising fermenting the first aqueous solution comprising glucose, the second aqueous solution comprising glucose, or a combination thereof, to produce a fermentation broth comprising ethanol.

66. The process according to claim 40, wherein about 75% to about 100% (w/w) of the total cellulase enzymes present bind to the fiber solids in the step of hydrolyzing (step (ii)).

67. The use of an enzyme composition in the enzymatic hydrolysis of cellulose to produce a hydrolysis product comprising glucose from an aqueous slurry of a pretreated lignocellulosic feedstock, the enzyme composition comprising cellulase enzymes, one or more than one β

-glucosidase enzyme and a binding agent for binding the β

-glucosidase enzyme to the pretreated lignocellulosic feedstock, wherein said enzymatic hydrolysis is performed in a solids-retaining hydrolysis reactor.