Enzymatic hydrolysis of lignocellulosic feedstocks using accessory enzymes

• US 8,017,361 B2
• Filed: 08/29/2008
• Issued: 09/13/2011
• Est. Priority Date: 08/30/2007
• Status: Expired due to Fees

• Current Assignee: Iogen Energy Corporation (Iogen Corporation)
• Original Assignee: Iogen Energy Corporation (Iogen Corporation)
• Inventors: Hill, Christopher, Tomashek, John, Scott, Brian R., Liu, Chengsong
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US12/201,374
• Publication Number:
  US 20090061484A1
• Time in Patent Office: 1,110 Days
• Field of Search: None
• US Class Current: 435/99
• 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 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: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• 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

Provided is an enzyme mixture for hydrolyzing a pretreated lignocellulosic feedstock to soluble sugars. The enzyme mixture comprises EG4 at a fractional concentration (f_EG4) of about 0.25 to about 0.83 (w/w), Swollenin at a fractional concentration (f_Swo1) of about 0 to about 0.66 (w/w), and Cip1 at a fractional concentration (f_Cip1) of 0 to about 0.33 measured relative to all accessory enzymes present in the enzyme mixture. Also provided are processes for converting a pretreated lignocellulosic feedstock to soluble sugars using the enzyme mixtures, and methods of using and producing such enzyme mixtures.

Claims

1. A cellulase enzyme mixture for hydrolyzing a pretreated lignocellulosic feedstock to soluble sugars, the cellulase enzyme mixture comprising EG4, Swollenin and Cip1 accessory enzymes, wherein

the EG4 accessory enzyme is present at a fractional concentration (f_EG4) of about 0.09 to about 0.91 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture,

the Swollenin accessory enzyme is present at a fractional concentration (f_Swo1) of about 0.09 to about 0.91 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture, and

the Cip1 accessory enzyme being present at a fractional concentration (f_Cip1) of 0 to about 0.42 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture.

2. The cellulase enzyme mixture of claim 1, wherein

the EG4 accessory enzyme is present at a fractional concentration (f_EG4) of about 0.25 to about 0.83 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture,

the Swollenin accessory enzyme is present at a fractional concentration (f_Swo1) of about 0.09 to about 0.66 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture, and

the Cip1 accessory enzyme being present at a fractional concentration (f_Cip1) of 0 to about 0.33 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture.

3. The cellulase enzyme mixture of claim 2, wherein the cellulase enzyme mixture comprises CBH1, CBH2, EG1 and EG2 primary cellulase enzymes.

4. The cellulase enzyme mixture of claim 3, wherein the primary cellulase enzymes have a combined content within the cellulase enzyme mixture of about 70 to about 95 weight percent measured relative to the primary cellulase enzymes and accessory enzymes present in the cellulase enzyme mixture, and wherein said accessory enzymes have a combined content within the cellulase enzyme mixture of about 5 to about 30 weight percent measured relative to the primary cellulase enzymes and accessory enzymes present in the cellulase enzyme mixture.

5. The cellulase enzyme mixture of claim 4, wherein the combined content of primary cellulase enzymes within the cellulase enzyme mixture measured relative to the primary cellulase enzymes and accessory enzymes present in the cellulase enzyme mixture is about 70 to about 90 weight percent, and wherein the combined content of accessory enzymes within the cellulase enzyme mixture measured relative to the primary cellulase enzymes and accessory enzymes present in the cellulase enzyme mixture is about 10 to about 30 weight percent.

6. The cellulase enzyme mixture of claim 3, wherein the primary cellulase enzymes and accessory enzymes have a combined content of about 70 to about 100 weight percent measured relative to total protein present in the cellulase enzyme mixture.

7. The cellulase enzyme mixture of claim 3, wherein the CBH1 and CBH2 enzymes have a combined content of about 55 to about 85 weight percent and the EG1 and EG2 enzymes have a combined content of about 15 to about 45 weight percent measured relative to the combined content of CBH1, CBH2, EG1 and EG2 enzymes present in the cellulase enzyme mixture.

8. The cellulase enzyme mixture of claim 7, wherein the CBH1 and CBH2 enzymes are each present at a fractional concentration of about 0.25 to about 0.75 (w/w) measured relative to the combined content of CBH1 and CBH2 enzymes present in the cellulase enzyme mixture.

9. The cellulase enzyme mixture of claim 7, wherein the EG1 and EG2 enzymes are each present at respective fractional concentrations of about 0.35 to about 0.95 (w/w) and about 0.05 to about 0.65 (w/w) measured relative to the combined content of EG1 and EG2 enzymes present in the cellulase enzyme mixture.

10. The cellulase enzyme mixture of claim 2, wherein said EG4 enzyme is present at a fractional concentration (f_EG4) of about 0.33 to about 0.50 (w/w) measured relative to all accessory enzymes present in said enzyme mixture.

11. The cellulase enzyme mixture of claim 2, wherein said Swollenin enzyme is present at a fractional concentration (f_Swo1) of about 0.33 to about 0.58 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture.

12. The cellulase enzyme composition of claim 2, wherein said Cip1 is present at a fractional concentration (f_Cip1) of about 0.08 to about 0.25 (w/w) measured relative to the EG4, Swollenin and Cip1 accessory enzymes present in the cellulase enzyme mixture.

13. The cellulase enzyme mixture of claim 2, wherein the enzyme mixture is a complete blend of secreted enzymes from a microbial source, the primary cellulase enzymes and accessory enzymes making up between about 70 and about 100 weight percent of the secreted enzymes in the blend and wherein the secreted enzymes comprise additional non-cellulase enzymes that make up between >

0 and 30 wt % of the secreted enzymes in the blend.

14. The cellulase enzyme mixture of claim 2, wherein the enzyme mixture comprises β

-glucosidase.

22. A process for converting a pretreated lignocellulosic feedstock to soluble sugars comprising the step of enzymatically hydrolyzing the pretreated lignocellulosic feedstock with the cellulase enzyme mixture according to claim 1.

23. The process of claim 22, wherein the EG4 accessory enzyme is present at a fractional concentration (f_EG4) of about 0.25-0.83 (w/w), said Swollenin accessory enzyme is present at a fractional concentration (f_Swo1) of about 0.09 to about 0.66 (w/w), and said Cip1 accessory enzyme is present at a fractional concentration (f_Cip1) of 0 to about 0.33 w/w) measured relative to all accessory enzymes present in said enzyme mixture.

24. The process of claim 22, wherein the cellulase enzyme mixture comprises CBH1, CBH2, EG1 and EG2 primary cellulase enzymes.

25. The process of claim 23, wherein the cellulase enzyme mixture comprises CBH1, CBH2, EG1 and EG2 primary cellulase enzymes.

26. The process of claim 25, wherein the primary cellulase enzymes have a combined content within the cellulase enzyme mixture of about 70 to about 95 weight percent measured relative to the primary cellulase enzymes and accessory enzymes present in the cellulase enzyme mixture, and wherein the accessory enzymes have a combined content within the cellulase enzyme mixture of about 5 to about 30 weight percent measured relative to the primary cellulase enzymes and accessory enzymes present in the cellulase enzyme mixture.

27. The process of claim 26, wherein the combined content of primary cellulase enzymes within the cellulase enzyme mixture measured relative to the primary cellulase enzymes and accessory enzymes present in the cellulase enzyme mixture is about 70 to about 90 weight percent and wherein the combined content within the cellulase enzyme mixture of accessory enzymes present in the cellulase enzyme mixture measured relative to the primary cellulase enzymes and accessory enzymes present in the cellulase enzyme mixture is about 10 to about 30 weight percent.

28. The process of claim 25, wherein the primary cellulase enzymes and accessory enzymes have a combined content within the cellulase enzyme mixture of about 70 to about 100 weight percent measured relative to total protein present in the cellulase enzyme mixture.

29. The process of claim 25, wherein the CBH1 and CBH2 enzymes have a combined content of about 55 to about 85 weight percent and the EG1 and EG2 enzymes have a combined content of about 15 to about 45 weight percent measured relative to CBH1, CBH2, EG1 and EG2 enzymes present in the cellulase enzyme mixture.

30. The process of claim 29, wherein the CBH1 and CBH2 enzymes are each present at a fractional concentration of about 0.25 to about 0.75 (w/w) measured relative to the combined content of CBH1 and CBH2 enzymes present in the cellulase enzyme mixture.

31. The process of claim 29, wherein the EG1 and EG2 enzymes are each present at respective fractional concentrations of from about 0.35 to about 0.95, (w/w) and from about 0.05 to about 0.65 (w/w) measured relative to the combined content of EG1 and EG2 enzymes present in the cellulase enzyme mixture.

32. The process of claim 23, wherein the EG4 enzyme is present at a fractional concentration (f_EG4) of about 0.33 to about 0.50 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture.

33. The process of claim 23, wherein the Swollenin enzyme is present at a fractional concentration (f_Swo1) of about 0.33 to about 0.58 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture.

34. The process of claim 23, wherein the Cip1 is present at a fractional concentration (f_Cip1) of about 0.08 to about 0.25 (w/w) measured relative to the EG4, Swollenin and Cip1 accessory enzymes present in the cellulase enzyme mixture.

35. The process of claim 25, wherein the primary cellulase enzymes and accessory enzymes are from a fungal source.

36. The process of claim 35, wherein the fungal source is an Ascomycete or Basidomycete fungus.

37. The process of claim 36, wherein the fungal source is selected from the group consisting of Trichoderma ssp., Aspergillus ssp., Hypocrea ssp., Humicola ssp., Neurospora ssp., Orpinomyces ssp., Gibberella ssp., Emericella ssp., Chaetomium ssp., Fusarium ssp., Penicillium ssp., Magnaporthe ssp., and Phanerochaete ssp.

38. The process of claim 37, wherein the fungal source is Trichoderma reesei.

39. The process of claim 25, wherein the primary cellulase enzymes and accessory enzymes are obtained from an organism by expressing coding sequences which are endogenous to the organism.

40. The process of claim 25, wherein the primary cellulase enzymes and accessory enzymes are obtained from an organism by expressing coding sequences which are heterologous to the organism.

41. The process of claim 25, wherein the primary cellulase enzymes and accessory enzymes are produced by expression in Trichoderma reesei.

42. The process of claim 23, wherein in the step of enzymatically hydrolyzing, at least 80 wt % of cellulose in the pretreated lignocellulosic feedstock is converted to soluble sugars.

43. The process of claim 42, wherein the step of enzymatically hydrolyzing is followed by a step of fermentation to produce ethanol, lactic acid, butanol, or a combination thereof from the soluble sugars.

44. The process of claim 25, wherein the cellulase enzyme mixture is a complete blend of secreted enzymes from a microbial source, the primary cellulase enzymes and accessory enzymes making up between about 70 and about 100 wt % of the secreted enzymes in the blend and wherein the secreted enzymes comprise additional non-cellulase enzymes that make up between >

0 and about 30 wt % of the secreted enzymes in the blend.

45. The process of claim 23, wherein the cellulase enzyme mixture comprises β

-glucosidase.

15. A cellulase enzyme mixture for hydrolyzing a pretreated lignocellulosic feedstock to soluble sugars, the cellulase enzyme mixture comprising EG4, Swollenin and Cip1 accessory enzymes, wherein

the EG4 accessory enzyme is present at a fractional concentration (f_EG4) of about 0.25 to about 0.83 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture,

the Swollenin accessory enzyme is present at a fractional concentration (f_Swo1) of about >

0 to about 0.66 (w/w) measured relative to all accessory enzymes present in the cellulase enzyme mixture, and

the Cip1 accessory enzyme being present at a fractional concentration (f_Cip1) of 0 to about 0.33 measured relative to all accessory enzymes present in the cellulase enzyme mixture.

17. A process for converting a pretreated lignocellulosic feedstock to soluble sugars comprising the step of enzymatically hydrolyzing the pretreated lignocellulosic feedstock with the cellulase enzyme mixture according to claim 15.

18. A method for producing the cellulase enzyme mixture according to claim 15, comprising the step of expressing the cellulase enzyme mixture from one or more microorganisms.

19. The method of claim 18, wherein the one or more microorganisms are genetically modified to adjust the levels of EG4, Swollenin, Cip1 or a combination thereof secreted by the one or more microorganisms.

20. The method of claim 19, wherein the one or more microorganisms are genetically modified by deleting one or more of the primary cellulase enzymes to adjust the expression levels of EG4, Swollenin, Cip1 or a combination thereof relative to the primary cellulases.

21. The method of claim 20, wherein the cellulase enzyme mixture is produced by conducting a fermentation at a pH of between about 2 and about 5 to adjust the expression levels of EG4, Swollenin, Cip1 or a combination thereof relative to one another or the expression levels of the accessory components relative to the primary cellulase enzymes.

16. A cellulase enzyme mixture for hydrolyzing a pretreated lignocellulosic feedstock to soluble sugars, the cellulase enzyme mixture comprising EG4, Swollenin and Cip1 accessory enzymes, wherein

the EG4 accessory enzyme is present at a fractional concentration (f_EG4) of about 0.25 to about 0.83 (w/w),

the Swollenin accessory enzyme is present at a fractional concentration (f_Swo1) of about 0.15 to about 0.66 (w/w), and

the Cip1 accessory enzyme being present at a fractional concentration (f_Cip1) of 0 to about 0.33 (w/w),

wherein each fractional concentration is measured relative to all of the EG4, Swollenin and Cip1 accessory enzymes present in the cellulase enzyme mixture.