Thermophilic mutants of Trichoderma reesei endoglucanase I

Patent metadata

Field	Value
Publication number	US-9410135-B2
Application number	US-201113822623-A
Country	US
Kind code	B2
Filing date	Aug 11, 2011
Priority date	Sep 15, 2010
Publication date	Aug 9, 2016
Grant date	Aug 9, 2016

Abstract

The present disclosure relates to mutant thermostable glycosyl hydrolases family 7 enzymes, including mutant Trichoderma reesei endoglucanase I. In particular, the present disclosure relates to mutant thermostable enzymes, compositions containing the enzymes, and methods of use thereof.

First claim

What is claimed is:

1. A mutant thermostable endoglucanase I enzyme, wherein the mutant thermostable enzyme has an amino acid substitution at position 230 of SEQ ID NO: 1 with an amino acid selected from the group consisting of
serine, threonine, leucine, methionine, lysine, alanine, glutamine, glutamic acid, and arginine; and
an amino acid substitution at position 113 of SEQ ID NO: 1 with an amino acid selected from the group consisting of leucine and serine.

2. A mutant thermostable endoglucanase I enzyme, wherein the mutant thermostable enzyme has
an amino acid substitution at position 230 of SEQ ID NO: 1 with an amino acid selected from the group consisting of serine, threonine, leucine, methionine, lysine, alanine, glutamine, glutamic acid, and arginine;
an amino acid substitution at position 113 of SEQ ID NO: 1 with an amino acid selected from the group consisting of leucine and serine; and
an amino acid substitution at position 115 of SEQ ID NO: 1 with an amino acid selected from the group consisting of threonine and glycine.

3. A mutant thermostable endoglucanase I enzyme, wherein the mutant thermostable enzyme has an amino acid substitution at position 230 of SEQ ID NO: 1 with an amino acid selected from the group consisting of serine, threonine, leucine, methionine, lysine, alanine, glutamine, glutamic acid, and arginine; and an amino acid substitution at position 115 of SEQ ID NO: 1 with an amino acid selected from the group consisting of threonine and glycine.

4. A mutant thermostable endoglucanase I enzyme, wherein the mutant thermostable enzyme has an amino acid substitution at position 113 of SEQ ID NO: 1 with an amino acid selected from the group consisting of leucine and serine; and an amino acid substitution at position 115 of SEQ ID NO: 1 with an amino acid selected from the group consisting of threonine and glycine.

5. The mutant thermostable enzyme of claim 1 , wherein the enzyme exhibits increased thermostability as compared to the wild-type enzyme after incubation at about 50° C. for about one hour.

6. The mutant thermostable enzyme of claim 1 , wherein the enzyme has a specific activity of at least about 0.5 mMole GE/μMole enzyme/hr after incubation at about 50° C. for about one hour.

7. A composition comprising the mutant thermostable enzyme of claim 1 .

8. An isolated nucleic acid encoding the mutant thermostable enzyme of claim 1 .

9. An expression vector comprising the nucleic acid of claim 8 operably linked to a regulatory sequence.

10. A host cell comprising the expression vector of claim 9 .

11. A composition comprising the host cell of claim 10 and culture medium.

12. A method of reducing the viscosity of a pretreated biomass mixture, comprising:
contacting a pretreated biomass mixture having an initial viscosity with the composition of claim 7 ; and
incubating the contacted biomass mixture under conditions sufficient to reduce the initial viscosity of said pretreated biomass mixture.

13. A method of converting biomass to sugars comprising contacting the biomass with the composition of claim 7 .

14. A method of hydrolyzing or degrading biomass, comprising contacting the biomass with the composition of claim 7 .

15. A method of producing a fermentation product, comprising:
contacting biomass with the composition of composition of claim 7 to form a first product; and
culturing the first product with one or more fermentative microorganisms or a chemical solution under conditions sufficient to produce a fermentation product.

16. The method of claim 15 , wherein the contacting is conducted at a temperature between about 50° C. and about 55° C., between about 55° C. and about 60° C., or between about 60° C. to about 70° C.

17. A method of fermenting biomass, comprising:
contacting biomass with one or more fermentative microorganisms, wherein the biomass is treated by the composition of claim 7 .

18. A method of producing a fuel comprising:
contacting biomass with the composition of claim 7 to yield a sugar solution; and
culturing the sugar solution with a fermentative microorganism or a chemical solution under conditions sufficient to produce a fuel.

19. The method of claim 18 , wherein the contacting is conducted at a temperature between about 50° C. and about 55° C., between about 55° C. and about 60° C., or between about 60° C. to about 70° C.

Assignees

• Chokhawala Harshal Akshay
• Kim Tae-Wan
• Blanch Harvey W
• Clark Douglas S
• Univ California

Inventors

• Chokhawala Harshal Akshay
• Kim Tae-Wan
• Blanch Harvey W
• Clark Douglas S

Classifications

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12P7/10

  substrate containing cellulosic material · CPC title

• Y02E50/16

  Cross-Sectional Technologies · mapped topic

• C12Y302/01004

  Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase · CPC title

• C12N9/2437 Primary

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title