Superactive Cellulase Formulation Using Cellobiohydrolase-1 from Penicillium Funiculosum

• US 20090081762A1
• Filed: 10/08/2008
• Published: 03/26/2009
• Est. Priority Date: 02/27/2003
• Status: Active Grant

• Current Assignee: Alliance For Sustainable Energy LLC
• Original Assignee: Alliance For Sustainable Energy LLC
• Inventors: Himmel, Michael E., Decker, Stephen R., Baker, John O., Chou, Yat-Chen, Adney, William S., Ding, Shi-You
• Granted Patent: US 8,283,150 B2
• Time in Patent Office: Days
• US Class Current: 435/209
• 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: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Abstract

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using purified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.

Claims

1. A cellulase formulation comprising:

a cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7)) enzyme having a polypeptide sequence of SEQ ID NO;

8; and

an endoglucanase present in amount ranging from one percent (1%) to twenty percent (20%) of the molar concentration of the Cel7 enzyme.

2. The cellulase formulation of claim 1, wherein the Cel7 enzyme is a heterologously expressed enzyme from a transgenic filamentous fungus.

3. The cellulase formulation of claim 2, wherein the transgenic filamentous fungus includes Aspergillus.

4. The cellulase formulation of claim 3, wherein the Aspergillus includes Aspergillus awamori.

5. The cellulase formulation of claim 2, wherein the transgenic filamentous fungus includes Trichoderma.

6. The cellulase formulation of claim 5, wherein the transgenic filamentous fungus includes Trichoderma reesei.

7. The cellulase formulation of claim 2, wherein the transgenic filamentous fungus includes Penicillium and a transgene coding for expression of the Cel7 enzyme is under control of a non-native promoter.

8. The cellulase formulation of claim 7, wherein the Penicillium comprises Penicillium funiculosum.

9. The cellulase formulation of claim 1, wherein the cellobiohydrolase formulation exhibits specific performance in saccharification of cellulose that exceeds by at least two-fold the specific performance of a comparable cellobiohydrolase formulation where Cel7A cellulase from Trichoderma reesei is substituted for the Cel7 enzyme.

10. The cellulase formulation of claim 9, wherein the specific performance is measured on corn stover.

11. The cellulase formulation of claim 1, wherein the endoglucanase comprises EIcd endoglucanase.

12. The cellulase formulation of claim 11, wherein the mixture comprises a 95:

5 molar ratio of the Cel7A enzyme to EIcd endoglucanase.

13. The cellulase formulation of claim 1, further comprising at least one additional hydrolyzing enzyme.

14. The cellulase formulation of claim 1, wherein the Cel7 enzyme is characterized by a peak activation temperature of 68°

C.

15. In a process for producing cellobiohydrolase 1 family 7 (Cel7) enzyme from a recombinant host, the improvement comprising:

transforming a filamentous fungus with a gene from Penicillium funiculosum, the gene encoding for a Cel7 enzyme foreign to the filamentous fungus; and

expressing the rCel7A enzyme from the filamentous fungus.

16. The process of claim 15, further comprising a step of purifying the Cel7 enzyme to a concentration than is obtainable from natural expression processes.

17. The process of claim 15 further comprising a step of saccharifying cellulose with the Cel7 enzyme.

18. A purified Cel7 enzyme produced by the process of claim 17.

19. A Cel7A enzyme produced by the process of claim 15.

20. A method of producing a recombinant cellobiohydrolase 1 (rCel7) enzyme, the method comprising the steps of:

obtaining a nucleic acid sequence that encodes a native Cel7A enzyme from Penicillium funiculosum.

transforming a filamentous fungus with the nucleic acid sequence; and

expressing rCel7A in the filamentous fungus by biological processes that operate on the nucleic acid sequence to produce rCel7 in the filamentous fungus.

21. The method of claim 20, wherein the filamentous fungus used in the transforming and expressing steps comprises Aspergillus.

22. The method of claim 21, wherein the Aspergillus comprises Aspergillus awamori.

23. The method of claim 20, wherein the filamentous fungus used in the transforming and expressing steps comprises Trichoderma.

24. The method of claim 23, wherein the Trichoderma comprises Trichoderma reesei.

25. The method of claim 24, further comprising a step of mixing the rCel7 enzyme with an endoglucanase.

26. The method of claim 25, wherein the endoglucanase comprises EIcd endoglucanase.

27. The method of claim 26, wherein the step of mixing comprises mixing a 95:

5 molar ratio of the rCel7A to EIcd endoglucanase.

28. A rCel7A produced by the method of claim 20.

29. A purified Cel7A cellulase according to SEQ ID NO:

8.

30. The purified Cel7A cellulase of claim 29 at least 50% pure.

31. The purified Cel7A cellulase of claim 29 at least 75% pure.

32. The purified Cel7A cellulase of claim 29 at least 90% pure.