Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

• US 8,283,150 B2
• Filed: 10/08/2008
• Issued: 10/09/2012
• Est. Priority Date: 02/27/2003
• Status: Active Grant

• Current Assignee: Alliance For Sustainable Energy LLC
• Original Assignee: Alliance For Sustainable Energy LLC
• Inventors: Adney, William S., Baker, John O., Decker, Stephen R., Chou, Yat-Chen, Himmel, Michael E., Ding, Shi-You
• Primary Examiner(s): Shukla, Ram R
• Assistant Examiner(s): Archie, Nina
• Application Number:
  US12/247,594
• Publication Number:
  US 20090081762A1
• Time in Patent Office: 1,462 Days
• Field of Search: 435/209, 435/185, 530/350
• 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 method of producing a recombinant cellobiohydrolase 1 (rCel7A) enzyme, wherein the rCel7A enzyme has the amino acid sequence of SEQ ID NO:

8, comprising;

obtaining a nucleic acid that encodes the amino acid sequence of SEQ ID NO 8 from Penicillium funiculosum, transforming a filamentous fungus with the nucleic acid; and

culturing the filamentous fungus under conditions that produce the rCel7A enzyme in the filamentous fungus.

2. The method of claim 1, wherein the filamentous fungus used in the transforming and expressing steps comprises Aspergillus.

3. The method of claim 2, wherein the Aspergillus comprises Aspergillus awamori.

4. The method of claim 1, wherein the filamentous fungus used in the transforming and expressing steps comprises Trichoderma.

5. The method of claim 4, wherein the Trichoderma comprises Trichoderma reesei.

6. The method of claim 1, further comprising a step of isolating the rCel7A enzyme.

7. The method of claim 6, further comprising a step of mixing the rCel7A enzyme with an endoglucanase.

8. The method of claim 7, wherein the endoglucanase comprises Elcd endoglucanase.

9. The method of claim 8, wherein the step of mixing comprises mixing a 95:

5 molar ratio of the rCel7A to Elcd endoglucanase.

12. A recombinant cellobiohydrolase 1 (rCel7A) enzyme from a filamentous fungus of the genus Penicillium, wherein the rCel7A enzyme has the amino acid sequence of SEQ ID NO:

8, wherein the rCel7A is produced by the method of claim 1.

10. A recombinant cellobiohydrolase 1 (rCel7A) enzyme having the amino acid sequence of SEQ ID NO:

8.

11. A recombinant cellobiohydrolase 1 (rCel7A) enzyme of claim 10, wherein the rCel7A enzyme is characterized by a peak activation temperature of 66.8°

C.