THERMOSTABLE CELLULASE AND METHODS OF USE

• US 20070256197A1
• Filed: 04/27/2007
• Published: 11/01/2007
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: C5-6 Technologies, LLC
• Original Assignee: C5-6 Technologies, Inc.
• Inventors: Brumm, Phillip
• Granted Patent: US 7,510,857 B2
• Time in Patent Office: Days
• US Class Current: 800/284
• CPC Class Codes: C12N 15/8246 Non-starch polysaccharides, e.g. cellulose, fructans, levans
• CPC Class Codes: C12N 9/2414 Alpha-amylase (3.2.1.1.)
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• 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 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/12 Disaccharides
• 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 19/18 produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12P 7/28 Acetone-containing products
• CPC Class Codes: C12P 7/56 Lactic acid
• CPC Class Codes: C12Y 302/01001 Alpha-amylase (3.2.1.1)
• CPC Class Codes: C12Y 302/01003 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01006 Endo-1,3(4)-beta-glucanase (3.2.1.6)
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

A Clostridium thermocellum thermostable cellulase enzyme with both endocellulase activity and exocellulase activity that is able to degrade cellulose in the absence of scaffolding and other cellulosomic proteins is provided. The use of the enzyme to degrade cellulosic materials to soluble sugars is also provided.

Claims

1. A purified thermostable cellulase comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:

1, wherein the cellulase exhibits endoglucanase or exoglucanase activity, or combinations thereof.

2. The cellulase of claim 1, wherein the cellulase is active in soluble form.

3. The cellulase of claim 1, comprising an amino acid sequence having at least about 95% identity to SEQ ID NO:

1.

4. The cellulase of claim 1, comprising the amino acid sequence shown in SEQ ID NO:

1.

5. The cellulase of claim 1, wherein the cellulase exhibits beta-glucanase, cellulase, activity, or combinations thereof.

6. The cellulase of claim 1, wherein the cellulase exhibits exo-cellulase activity.

7. A polynucleotide construct, comprising a polynucleotide encoding the cellulase of claim 1 operably connected to a promoter.

8. The polynucleotide construct of claim 7, wherein the promoter is a heterologous promoter.

9. A recombinant host cell, comprising the polynucleotide construct of claim 7.

10. A recombinant plant cell, comprising the polynucleotide construct of claim 7.

11. A transgenic plant comprising the recombinant plant cell of claim 10.

12. A composition comprising the cellulase of claim 1.

13. The composition of claim 12, further comprising an enzyme selected from a β

-glucosidase, an alpha-amylase, or a glucoamylase, or combinations thereof.

14. A method of producing at least one cellulose byproduct comprising contacting a cellulosic material with the cellulase of claim 1.

15. The method of claim 14, wherein a first byproduct is cellobiose.

16. The method of claim 15, further comprising contacting the cellobiose with a β

-glucosidase to produce glucose.

17. The method of claim 16, wherein the β

-glucosidase is thermostable.

18. The method of claim 16, further comprising fermenting the glucose to produce a second byproduct.

19. The method of claim 18, wherein the second byproduct is ethanol, lactic acid, or acetone.

20. The method of claim 14, wherein the method comprises contacting the cellulosic material with the cellulase at a temperature of about 40°

C. to about 70°

C.

21. The method of claim 14, wherein the method comprises contacting the cellulosic material with the cellulase at a pH of about 4.0 to about 6.0.

22. A method of producing ethanol comprising:

a) contacting a cellulosic material with the cellulase of claim 1 to produce cellobiose;

b) contacting the cellobiose with a β

-glucosidase to produce glucose; and

c) fermenting the glucose to produce ethanol.

23. The method of claim 22, wherein the cellulosic material is a plant material.

24. The method of claim 22, wherein the plant material is selected from the group consisting of wood, corn, sorghum, barley, wheat, oat, rice and cotton.

25. The method of claim 22, wherein the cellulosic material is a paper or a textile.