Glycoside hydrolases having multiple hydrolase activities

Patent metadata

Field	Value
Publication number	US-9725749-B2
Application number	US-201214114990-A
Country	US
Kind code	B2
Filing date	May 1, 2012
Priority date	May 2, 2011
Publication date	Aug 8, 2017
Grant date	Aug 8, 2017

Abstract

Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

First claim

What is claimed is:

1. An isolated recombinant glycoside hydrolase that is a variant of a native GH5 A4 subfamily glycoside hydrolase, wherein the variant glycoside hydrolase:
(i) has increased cellulase and/or mannanase activity compared to the native GH5 A4 subfamily glycoside hydrolase,
(ii) has at least 95% amino acid sequence identity to the native GH5 A4 subfamily glycoside hydrolase, and
(iii) comprises the following amino acids at positions in the native GH4A4 subfamily glycoside hydrolase that correspond to positions 20, 23, 53, 95, 96, and 287 of SEQ ID NO:1:
asparagine at position 20,
aspartic acid or glutamic acid at position 23,
proline at position 53,
histidine at position 95,
histidine at position 96 and
aspartic acid or glutamic acid at position 287,
wherein at least one of the amino acid residues at position 20, 23, 53, 95, 96, and 287 is substituted for another amino acid as compared to the amino acid that is present at that position in the native GH4A4 subfamily glycoside hydrolase.

2. The recombinant glycoside hydrolase of claim 1 , wherein the recombinant glycoside hydrolase has a cellulase activity and/or mannanase activity at least 10% higher than the activity of glycoside hydrolase of the native GH5 A4 subfamily glycoside hydrolase.

3. The recombinant glycoside hydrolase of claim 1 , wherein the variant glycoside hydrolase has cellulase activity, xylanase activity, and mannanase activity.

4. The recombinant glycoside hydrolase of claim 1 , wherein the variant glycoside hydrolase has at least 95% amino acid sequence identity to any one of SEQ ID NOS:5, 7, or 9.

5. A method of generating free sugars from a mixture comprising cellulose and mannan, the method comprising contacting the mixture with a glycoside hydrolase of claim 1 under conditions such that the glycoside hydrolase hydrolyzes the at least two polymeric substrates, thereby generating free sugars.

6. The method of claim 5 , wherein the mixture comprises cellulose, xylan and mannan and the glycoside hydrolase hydrolyzes each of the cellulose, xylan and mannan.

7. The method of claim 5 , wherein the variant glycoside hydrolase has at least 95% amino acid sequence identity to any one of SEQ ID NOS:5, 7, or 9.

8. The recombinant glycoside hydrolase of claim 1 , wherein the variant glycoside hydrolase has at least 95% identity to SEQ ID NO:9.

Assignees

• Chen Zhiwei
• Friedland Gregory D
• Chhabra Swapnil R
• Chivian Dylan C
• Simmons Blake A
• Univ California
• Sandia Corp

Inventors

• Chen Zhiwei
• Friedland Gregory D
• Chhabra Swapnil R
• Chivian Dylan C
• Simmons Blake A

Classifications

• C12Y302/01004

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

• C12N9/2437

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

• C12N9/2434 Primary

  acting on beta-1,4-glucosidic bonds · CPC title

• C12P19/14 Primary

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

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title