Endoglucanase mutants and mutant hydrolytic depolymerizing enzymes and uses thereof

• US 20030054535A1
• Filed: 11/19/2001
• Published: 03/20/2003
• Est. Priority Date: 05/19/1999
• Status: Abandoned Application

• Current Assignee: Midwest Research Institute
• Original Assignee: Midwest Research Institute
• Inventors: Thomas, Steven R., Himmel, Michael E., Adney, William S., Vinzant, Todd B., Sakon, Joshua, Decker, Stephen R., Baker, John O.
• Application Number:
  US09/997,504
• Publication Number:
  US 20030054535A1
• 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: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The invention provides a method for increasing the specific activity of a glycosyl hydrolase on a substrate, comprising replacing a hydrophobic surface binding amino acid of the hydrolase with a positively charged amino acid; and a method for increasing the specific activity of a glycosyl hydrolase on a substrate, comprising replacing an active site associated glycosyl-stabilizing amino acid of the hydrolase with an amino acid, the replacing amino acid not strongly retarding cellobiose from leaving the active site. The invention further provides mutant glycosyl hydrolases, which include Y245G, Y42R, and W82R.

Claims

1. A method for increasing the specific activity of a glycosyl hydrolase on a substrate, comprising replacing a hydrophobic surface binding amino acid of the hydrolase with a positively charged amino acid, to provide a mutant glycosyl hydrolase,

2. The method of claim 1, wherein the hydrophobic surface binding amino acid includes tryptophan or tyrosine and the positively charged amino acid is arginine.

5. The methods of claims 1 and 3 wherein replacing comprises site-directed-mutagenesis.

6. The methods of claims 1 and 3 wherein the mutant glycosyl hydrolase comprises a mutant EI endoglucanase.

7. The methods of claims 1 and 3 wherein the mutant glycosyl hydrolase comprises Y245G, Y42R, W82R, or a mixture thereof.

8. The methods of claims 1 and 3, wherein the substrate comprises pretreated biomass.

3. A method for increasing the specific activity of a glycosyl hydrolase on a substrate, comprising replacing an active site associated glycosyl-stabilizing amino acid of the hydrolase with an amino acid, the replacing amino acid not strongly retarding cellobiose from leaving the active site to provide a mutant glycosyl hydrolase.

4. The method of claim 3, wherein the glycosyl-stabilizing amino acid comprises tyrosine 3 and the replacing amino acid comprises glycine.

9. A mutant glycosyl hydrolase having enhanced catalytic activity, said mutant glycosyl hydrolase comprising an amino having a positively charged amino acid at a position occupied by a hydrophobic surface binding amino acid in a wild-type glycosyl hydrolase amino acid sequence, wherein said mutant glycosyl hydrolase has an enhanced catalytic activity of 10% to 50% compared to catalytic activity of the wild-type glycosyl hydrolase.

10. The mutant glycosyl hydrolase of claim 9 further defined as a cellulase.

11. The mutantglycosyl hydrolase of claim 9 further defined as Y245G.

12. The mutant glycosly hydrolase of claim 9 further defined as Y42R.

13. The mutant glycosyl hydrolase of claim 9 further defined as a mannanase.

14. The mutant glycosyl hydrolase of claim 9 further defined as comprising W82R.

15. A method for converting a biomass into ethanol comprising:

a. mixing a composition comprising biomass with a mutant glycosyl hydrolase having enhanced catalytic activity over a wild-type glycosyl hydrolase to provide a soluble fermentable sugar preparation; and

b. fermenting said soluble fermentable sugar preparation to provide a composition comprising ethanol.

16. The method of claim 15 wherein the biomass is a cellulosic biomass.

17. The method of claim 15 wherein the mutant glycosyl hydrolase is Y245G.

18. The method of claim 15 wherein the mutant glycosyl hydrolase is Y82R.

19. The method of claim 15 wherein the mutant glycosyl hydrolase is W42R.

20. The method of claim 15 wherein the mutant glycosyl hydrolase comprises Y245G, Y82R, or W42R.

21. The method of claim 15 wherein the biomass is further admixed with a glycohydrolase.

22. A method for converting a biomass to a lactic acid comprising:

a. admixing a composition comprising a biomass with a glycosyl mutant hydrolase having enhanced catalytic activity over a wild-type glycosyl hydrolase to provide a soluble fermentable sugar preparation; and

b. fermenting said soluble fermentable sugar preparation to provide lactic acid.

23. The method of claim 22 wherein the lactic acid is further defined as a monomer feedstock for production of biodegradable plastics.

24. The method of claim 22 wherein the mutant glycosyl hydrolase is Y82R, W42R, Y245G, or a mixture thereof.

25. The method of claim 22 wherein the glycosyl hydrolase mutant is mutant Y82R, W42R, Y245G, or a mixture thereof.

26. A method for increasing the specific activity of a hydrolytic depolymerizing enzyme, comprising replacing an extended-active site residue that binds strongly to the leaving group with another that binds much less strongly to the leaving group.