Thermophilic cellobiohydrolase
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.
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.