Methods of hydrolyzing a cellulose using halophilic, thermostable and ionic liquids tolerant cellulases

Patent metadata

Field	Value
Publication number	US-9862982-B2
Application number	US-201615164813-A
Country	US
Kind code	B2
Filing date	May 25, 2016
Priority date	Jun 10, 2011
Publication date	Jan 9, 2018
Grant date	Jan 9, 2018

Abstract

The present invention provides for an isolated or recombinant polypeptide comprising an amino acid sequence having at least 70% identity with the amino acid sequence of a Halorhabdus utahensis cellulase, such as Hu-CBH1, wherein said amino acid sequence has a halophilic thermostable and/or thermophilic cellobiohydrolase (CBH) activity. In some embodiments, the polypeptide has a CBH activity that is resistant to up to about 20% of ionic liquids. The present invention also provides for compositions comprising and methods using the isolated or recombinant polypeptide.

First claim

What is claimed is:

1. A method of hydrolyzing a cellulose, comprising: (a) providing a composition comprising (i) an isolated or recombinant polypeptide comprising an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:5, wherein said isolated or recombinant polypeptide comprises a catalytic domain, a fibronectin domain 3 (FN-3) and an Ig-like domain, and has a halophilic thermostable or thermophilic cellobiohydrolase (CBH) activity, (ii) a suitable salt concentration, (iii) an ionic liquid (IL), and (iv) a cellulose; and (b) incubating the composition for a suitable length of time, such that the cellulose is hydrolyzed by the polypeptide.

2. The method of claim 1 , wherein the composition comprises a pretreatment biomass.

3. The method of claim 1 , wherein the amino acid sequence has at least 95% identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:5.

4. The method of claim 3 , wherein the amino acid sequence has at least 99% identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:5.

5. The polypeptide of claim 4 , wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:5.

6. The method of claim 2 , wherein the composition has a temperature of from about 37° C. to about 90° C.

7. The method of claim 6 , wherein the composition has a temperature of from about 50° C. to about 90° C.

8. The method of claim 7 , wherein the composition has a temperature of from about 60° C. to about 90° C.

9. The method of claim 2 , wherein the composition has a NaCl, or other suitable salt, or mixture thereof, concentration of from about 0.1 M to about 5 M.

10. The method of claim 9 , wherein the composition has a NaCl, or other suitable salt, or mixture thereof, concentration of from about 2 M to about 5 M.

11. The method of claim 2 , wherein the composition has a pH of from 7.5 to 12.5.

12. The method of claim 11 , wherein the composition has a pH of equal to or more than about 8 pH.

13. The method of claim 2 , wherein the composition has a concentration of IL equal to or more than 1% w/w.

14. The method of claim 13 , wherein the composition has a concentration of IL of 20 to 30%.

15. The method of claim 2 , wherein the composition comprises 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.

16. The method of claim 15 , wherein the composition comprises 1-allyl-3-methylimidazolium acetate (AMIM Acetate), 1-allyl-3-methylimidazolium chloride (AMIM Cl), 1-allyl-3-methylimidazolium hydrogensulfate (AMIM HOSO 3 ), 1-allyl-3-methylimidazolium methylsulfate (AMIM MeOSO 3 ), 1-allyl-3-methylimidazolium ethylsulfate (AMIM EtOSO 3 ), 1-allyl-3-methylimidazolium methanesulfonate (AMIM MeSO 3 ), 1-allyl-3-methylimidazolium tetrachloroaluminate (AMIM AlCl 4 ), 1-ethyl-3-methylimidazolium acetate (EMIM Acetate), 1-ethyl-3-methylimidazolium chloride (EMIM 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, choline salicylate, or a mixture thereof.

17. The method of claim 2 , wherein the incubating step results in at least 50% hydrolysis of the cellulose into cellobioses.

18. The method of claim 17 , wherein the incubating step results in at least 70% hydrolysis of the cellulose into cellobioses.

Assignees

• Univ California
• Sandia Corp

Inventors

• Zhang Tao
• Datta Supratim
• Simmons Blake A
• Rubin Edward M

Classifications

• 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

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P19/02

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

• C12N9/2437

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