GH61 glycoside hydrolase protein variants and cofactors that enhance GH61 activity

• US 8,951,758 B2
• Filed: 08/22/2012
• Issued: 02/10/2015
• Est. Priority Date: 08/22/2011
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Yang, Jie, Zhang, Xiyun, Yoon, Jungjoo, Rao, Kripa, Grate, John H., Elgart, David, Baidyaroy, Dipnath
• Primary Examiner(s): KAM, CHIH MIN
• Application Number:
  US13/592,060
• Publication Number:
  US 20130052698A1
• Time in Patent Office: 902 Days
• Field of Search: 435/188, 435/196, 435/198, 435/320.1, 435/254.11, 435/254.2, 435/99, 435/139, 435/136, 435/144, 536/23.2
• US Class Current: 435/99
• CPC Class Codes: C12N 9/242 Fungal source
• CPC Class Codes: C12N 9/2434 acting on beta-1,4-glucosidic bonds
• 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: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention provides various GH61 protein variants comprising various amino acid substitutions. The GH61 protein variants have an improved ability to synergize with cellulase enzymes, thereby increasing the yield of fermentable sugars obtained by saccharification of biomass. In some embodiments, sugars obtained from saccharification are fermented to produce numerous end-products, including but not limited to alcohol.

Claims

1. A method of producing at least one fermentation end product from at least one cellulosic substrate, comprising:

a) providing at least one cellulosic substrate and at least one enzyme composition comprising at least one glycoside hydrolase 61 (GH61) variant protein comprising the amino acid sequence of SEQ ID NO;

5, having GH61 activity;

b) contacting the cellulosic substrate with the enzyme composition under conditions whereby fermentable sugars are produced from the cellulosic substrate in a saccharification reaction; and

c) contacting the fermentable sugars with a microorganism capable of fermenting said fermentable sugars under fermentation conditions such that at least one fermentation end product is produced.

2. The method of claim 1, wherein said method comprises simultaneous saccharification and fermentation reactions (SSF).

3. The method of claim 1, wherein saccharification of the cellulosic substrate and said fermentation in separate reactions (SHF).

4. The method of claim 1, further comprising at least one adjunct material in said saccharification reaction.

5. The method of claim 4, wherein said adjunct material is selected from at least one divalent metal cation, gallic acid, and/or at least one surfactant.

6. The method of claim 1, wherein said method is conducted at about pH 5.0 or about pH 6.0.

7. The method of claim 1, further comprising recovering at least one end product.

8. The method of claim 1, wherein said fermentation end product is fermentation end product is selected from alcohols, fatty alcohols, fatty acids, lactic acid, acetic acid, 3-hydroxypropionic acid, acrylic acid, succinic acid, citric acid, malic acid, fumaric acid, amino acids, 1,3-propanediol, ethylene, glycerol, butadiene, and/or beta-lactams.

9. The method of claim 8, wherein said fermentation end product is an alcohol selected from ethanol and butanol.

10. The method of claim 9, wherein said alcohol is ethanol.