Thermophilic cellobiohydrolase

Patent metadata

Field	Value
Publication number	US-9624482-B2
Application number	US-89272410-A
Country	US
Kind code	B2
Filing date	Sep 28, 2010
Priority date	Sep 28, 2009
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

The present invention provides for a composition comprising a polypeptide comprising a first amino acid sequence having at least 70% identity with the amino acid sequence of Csac GH5 wherein said first amino acid sequence has a thermostable or thermophilic cellobiohydrolase (CBH) or exoglucanase activity.

First claim

What is claimed is:

1. A composition comprising (a) a polypeptide comprising a first amino acid sequence comprising the amino acid sequence of amino acids 636-975 of Caldicellulosiruptor saccharolyticus (“Csac”) cellobiohydrolase (CBH) wherein said first amino acid sequence has a thermostable or thermophilic CBH or exoglucanase activity and the Csac CBH has the amino acid sequence of SEQ ID NO:2; and (b) more than 0% to 50% of an ionic liquid (“IL”).

2. The composition of claim 1 , wherein the polypeptide further comprises a second amino acid sequence having at least 90% identity with the amino acid sequence of amino acids 424-506 of SEQ ID NO:2 (carbohydrate binding module family 3 (CBM3)) wherein said second amino acid sequence is capable of binding a carbohydrate.

3. The composition of claim 2 , wherein the carbohydrate is a cellulose.

4. The composition of claim 3 , wherein the composition further comprises a cellulose capable of being cleaved by a Csac glycoside hydrolase family 5 (GH5) to produce a cellobiose.

5. The composition of claim 1 , wherein the composition has a temperature of at least 65° C.

6. The composition of claim 5 , wherein the composition has a temperature of at least 80° C.

7. The composition of claim 1 , wherein the composition has a pH from 4.7 to 5,5.

8. The composition of claim 1 , wherein the composition comprises more than 0% to 35% of the ionic liquid (“IL”).

9. The composition of claim 2 , wherein the second amino acid sequence has at least 95% identity with amino acids 424-506 of SEQ ID NO:2.

10. The composition of claim 9 , wherein the second amino acid sequence has at least 99% identity with amino acids 424-506 of SEQ ID NO:2.

11. The composition of claim 10 , wherein the second amino acid sequence comprises amino acids 424-506 of SEQ ID NO:2.

12. The composition of claim 1 , wherein the composition further comprises one or more thermostable or thermophilic endoglucanase enzymes.

13. A method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising a solution comprising more than 0% to 50% of an ionic liquid (“IL”) and a cellulose, and (b) introducing a polypeptide comprising an amino acid sequence comprising the amino acid sequence of amino acids 636-975 of Caldicellulosiruptor saccharolyticus (“Csac”) cellobiohydrolase (CBH) wherein said amino acid sequence has a thermostable or thermophilic CBH or exoglucanase activity and the Csac CBH has the amino acid sequence of SEQ ID NO:2, such that the cellulose is hydrolyzed by the polypeptide.

14. The method of claim 13 wherein the solution comprises a pretreatment biomass comprising the cellulose.

15. The method of claim 14 , wherein the pretreatment biomass is a pretreatment cellulose biomass, pretreatment hemicellulose biomass, pretreatment lingo-cellulose biomass, or a mixture thereof.

16. The method of claim 13 , wherein the IL is 1-alkyl-3-alkylimidazolium alkanate, 1-alkyl-3-alkylimidazolium alkylsulfate, 1-alkyl-3-alkylimidazolium methylsulfonate, 1-alkyl-3-alkylimidazolium hydrogensulfate, 1-alkyl-3-alkylimidazolium thiocyanate, or 1-alkyl-3-alkylimidazolium halide, wherein an “alkyl” is an alkyl group comprising from 1 to 10 carbon atoms, and an “alkanate” is an alkanate comprising from 1 to 10 carbon atoms.

17. The method of claim 13 , wherein the IL is 1-ethyl-3-methylimidazolium acetate (EMIN Acetate), 1-ethyl-3-methylimidazolium chloride (EMIN Cl), 1-ethyl-3-methylimidazolium hydrogensulfate (EMIM HOSO 3 ), 1-ethyl-3-methylimidazolium methylsulfate (EMIM MeOSO 3 ), 1-ethyl-3-methylimidazolium ethylsulfate (EMIM EtOSO 3 ), 1-ethyl-3-methylimidazolium methanesulfonate (EMIM MeSO 3 ), 1-ethyl-3-methylimidazolium tetrachloroaluminate (EMIM AlCl 4 ), 1-ethyl-3-methylimidazolium thiocyanate (EMIM SCN), 1-butyl-3-methylimidazolium acetate (BMIM Acetate), 1-butyl-3-methylimidazolium chloride (BMIM Cl), 1-butyl-3-methylimidazolium hydrogensulfate (BMIM HOSO 3 ), 1-butyl-3-methylimidazolium methanesulfonate (BMIM MeSO 3 ), 1-butyl-3-methylimidazolium methylsulfate (BMIM MeOSO 3 ), 1-butyl-3-methylimidazolium tetrachloroaluminate (BMIM AlCl 4 ), 1-butyl-3-methylimidazolium thiocyanate (BMIM SCN), 1-ethyl-2,3-dimethylimidazolium ethylsulfate (EDIM EtOSO 3 ), Tris(2-hydroxyethyl)methylammonium methylsulfate (MTEOA MeOSO 3 ), 1-methylimidazolium chloride (MIM Cl), 1-methylimidazolium hydrogensulfate (MIM HOSO 3 ), 1,2,4-trimethylpyrazolium methylsulfate, tributylmethylammonium methylsulfate, choline acetate, or choline salicylate.

18. The method of claim 13 , wherein the IL comprises a concentration equal to or more than 1% of the solution.

19. The method of claim 18 , wherein the IL comprises a concentration equal to or more than 5% of the solution.

20. The method of claim 19 , wherein the IL comprises a concentration equal to or more than 10% of the solution.

21. The method of claim 20 , wherein the IL comprises a concentration equal to or more than 20% of the solution.

22. The method of claim 1 , wherein the solution has a temperature of at least 65° C.

23. The method of claim 22 , wherein the solution has a temperature of at least 80° C.

24. The method of claim 1 , wherein the solution has a pH from 4.7 to 5.5.

25. The composition of claim 1 , wherein the concentration of IL in the composition is equal to or more than 1%.

26. The composition of claim 25 , wherein the concentration of IL in the composition is equal to or more than 10%.

27. The composition of claim 1 , wherein the IL is 1-alkyl-3-alkylimidazolium alkanate, 1-alkyl-3-alkylimidazolium alkylsulfate, 1-alkyl-3-alkylimidazolium methylsulfonate, 1-alkyl-3-alkylimidazolium hydrogensulfate, 1-alkyl-3-alkylimidazolium thiocyanate, or 1-alkyl-3-alkylimidazolium halide, wherein an “alkyl” is an alkyl group comprising from 1 to 10 carbon atoms, and an “alkanate” is an alkanate comprising from 1 to 10 carbon atoms.

28. The composition of claim 1 , wherein the IL is 1-ethyl-3-methylimidazolium acetate (EMIN Acetate), 1-ethyl-3-methylimidazolium chloride (EMIN Cl), 1-ethyl 3-methylimidazolium hydrogensulfate (EMIM HOSO 3 ), 1-ethyl-3-methylimidazolium methylsulfate (EMIM MeOSO 3 ), 1-ethyl-3-methylimidazolium ethylsulfate (EMIM EtOSO 3 ), 1-ethyl-3-methylimidazolium methanesulfonate (EMIM MeSO 3 ), 1-ethyl-3-methylimidazolium tetrachloroaluminate (EMIM AlCl 4 ), 1-ethyl-3-methylimidazolium thiocyanate (EMIM SCN), 1-butyl-3-methylimidazolium acetate (BMIM Acetate), 1-butyl-3-methylimidazolium chloride (BMIM Cl), 1-butyl-3-methylimidazolium hydrogensulfate (BMIM HOSO 3 ), 1-butyl-3-methylimidazolium methanesulfonate (BMIM MeSO 3 ), 1-butyl-3-methylimidazolium methylsulfate (BMIM MeOSO 3 ), 1-butyl-3-methylimidazolium tetrachloroaluminate (BMIM AlCl 4 ), 1-butyl-3-methylimidazolium thiocyanate (BMIM SCN), 1-ethyl-2,3-dimethylimidazolium ethylsulfate (EDIM EtOSO 3 ), Tris(2-hydroxyethyl)methylammonium methylsulfate (MTEOA MeOSO 3 ), 1-methylimidazolium chloride (MIM Cl), 1-methylimidazolium hydrogensulfate (MIM HOSO 3 ), 1,2,4-trimethylpyrazolium methylsulfate, tributylmethylammonium methylsulfate, choline acetate, or choline salicylate.

29. The composition of claim 1 , wherein the polypeptide further comprises a C-terminal tag.

30. The composition of claim 29 , wherein the C-terminal tag is a V5 epitope tag or a His tag.

Assignees

• Sapra Rajat
• Park Joshua I
• Datta Supratim
• Simmons Blake A
• Sandia Corp

Inventors

• Sapra Rajat
• Park Joshua I
• Datta Supratim
• Simmons Blake A

Classifications

• C12N9/2437 Primary

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

• 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

• C12Y302/01091

  Cellulose 1,4-beta-cellobiosidase (3.2.1.91) · CPC title

• C12P2201/00

  Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis · CPC title

• C12P2203/00

  Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source · CPC title