Method for glucose production using endoglucanase core protein for improved recovery and reuse of enzyme

• US 20060008885A1
• Filed: 03/05/2003
• Published: 01/12/2006
• Est. Priority Date: 03/15/2002
• Status: Active Grant

• Current Assignee: Iogen Energy Corporation (Iogen Corporation)
• Original Assignee: Iogen Energy Corporation (Iogen Corporation)
• Inventors: Tolan, Jeffrey S., White, Theresa C., Donaldson, Jennifer, Wahnon, Daphne
• Granted Patent: US 8,012,721 B2
• Time in Patent Office: Days
• US Class Current: 435/105
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• 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: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention pertains to a method of converting cellulose to glucose by treating a pretreated lignocellulosic substrate with an enzyme mixture comprising cellulase enzyme and endoglucanase core proteins, wherein the endoglucanase core proteins are present in the enzyme mixture at an amount relative to all endoglucanases from about 35 wt. % to about 100 wt. % and wherein the endoglucanase cellulase enzymes are present in the enzyme mixture at an amount relative to the amount of CBH and EG enzymes from about 2 wt. % to about 50 wt. %. The pretreated lignocellulosic substrate is selected from the group consisting of agricultural residues, residues after starch or sugar removal, dedicated ethanol crops, forestry products, and pulp and paper products, or combinations thereof.

Claims

1. A method of converting cellulose to glucose comprising, treating a pretreated lignocellulosic substrate in an enzyme mixture comprising cellobiohydrolase (CBH), β

-glucosidase, and endoglucanase (EG) enzymes, for a sufficient time for production of said glucose, wherein said EG enzymes comprise from about 2 wt % to about 50 wt % of the CBH and EG enzymes present, and said EG type enzyme comprises from about 35 wt % to about 100 wt % endoglucanase core protein (ECP).

2. The method of claim 1 wherein EG enzymes comprise 7 wt % to 30 wt % of the CBH and EG enzymes present.

3. The method of claim 1 wherein said ECP is from about 50 wt % to about 90 wt % of said EG type enzyme.

4. The method of claim 3 wherein said ECP is from about 60 wt % to about 80 wt % of said EG type enzyme.

5. The method of claim 1, wherein said ECP is selected from the group consisting of EGI core, EGII core, EGI with inactivated cellulose binding domain, EGH with inactivated cellulose binding domain, EGI core plus linker, EGII core plus linker, and a combination thereof.

6. The method of claim 1, wherein the CBH, β

-glucosidase, and EG enzymes are obtained from Trichoderma, Hypocrea, Thermomonospora, Aspergillus, Streptomyces, Cellulomonas, Humicola, Fusarium, Fibrobacter, Clostridium, Bacillus, or a combination thereof.

7. The method of claim 6, wherein said CBH and EG enzymes are obtained from Trichoderma, and said β

-glucosidase is obtained from Aspergillus or Trichoderma

8. The method of claim 7, wherein said CBH and EG enzymes are from Trichoderma reesei.

9. The method of claim 1, wherein following said step of treating, is a step of recovering said ECP.

10. The method of claim 9, wherein said ECP is reused following said step of recovering.

11. The method of claim 1, wherein said pretreated lignocellulosic substrate is selected from the group consisting of agricultural residues, residues after starch or sugar removal, dedicated ethanol crops, forestry products, and pulp and paper products or combinations thereof.

12. The method of claim 11 wherein,

said agricultural residues are selected from the group consisting of corn stover, wheat straw, barley straw, and soybean stover;

said residues after starch or sugar removal, are selected from the group consisting of oat hulls, rice hulls, sugar cane bagasse, and corn fibre;

said dedicated ethanol crops are selected from the group consisting of switch grass, miscanthus, cord grass, and rye grass;

said forestry products are selected from the group consisting of hardwood, softwood, Eucalyptus, and sawdust; and

said pulp and paper products is solka floc.

15. The method of claim 1, wherein the pretreated lignocellulosic substrate that is being treated with said enzyme mixture is present at a concentration of about 1 wt % to about 25 wt %.

16. The method of claim 12, wherein said lignocellulosic substrate that is being treated with said enzyme mixture is present at a concentration of about 10 wt % to about 16 wt %.

17. The method of claim 9, wherein said step of recovering, comprises precipitating said ECP, or using an ultrafiltration membrane to recover said ECP from solution.

18. The method of claim 1, wherein said pretreated lignocellulosic substrate is pretreated using an acid steam cook.

22. The method of claim 1, wherein said CBH enzymes comprise Cellobiohydrolase I (CBHI) and Cellobiohydrolase II (CBHII).

23. The method of claim 22, wherein CBHI is present from about 25 wt % to about 95 wt % of said CBH enzymes.

24. The method of claim 22 wherein the CBUH is from Trichoderma.

25. The method of claim 22 wherein the CBI is from Trichoderma.

13. A method of converting cellulose to glucose comprising treating a pretreated lignocellulosic substrate with an enzyme mixture comprising cellulase enzyme with an EG core protein (ECP), for a period of time sufficient to hydrolyze said cellulose;

and recovering said ECP from said enzyme mixture.

14. The method of claim 13, wherein said ECP is selected from the group consisting of EGI core, EGI core plus linker, EGI with inactivated cellulose binding domain, EGII core, EGII core plus linker, EGII with inactivated cellulose binding domain, and a combination thereof.

19. A method for converting cellulose to glucose comprising treating a pretreated lignocellulosic substrate with an enzyme mixture comprising cellobiohydrolase (CBH), β

-glucosidase, endoglucanase (EG), and EG-core plus linker enzyme, wherein said EG-core plus linker enzyme is present in an amount of about 2 wt % to about 50 wt %, relative to the amounts of said CBH and EG enzyme.

20. The method of claim 19, wherein said amount of EG-core plus linker in said enzyme mixture is from about 7 wt % to about 50 wt %.

21. The method of claim 19, wherein said amount of EG-core plus linker in said enzyme mixture is from about 7 wt % to about 30 wt %.

26. A method for converting cellulose to glucose comprising treating a pretreated lignocellulosic substrate with an enzyme mixture comprising cellobiohydrolase (CBH, β

-glucosidase, endoglucanase (EG), and EG-core enzyme, wherein said EG-core enzyme is present in said enzyme mixture in an amount of about 2 wt % to about 50 wt %, relative to the amount of said CBH and EG enzymes.

27. The method of claim 26, wherein said amount of EG-core enzyme in said enzyme mixture is from about 7 wt % to about 50 wt %.

28. The method of claim 27, wherein said amount of EG-core enzyme in said enzyme mixture is from about 7 wt % to about 30 wt %.

29. A method for converting cellulose to glucose comprising treating a pretreated lignocellulosic substrate with an enzyme mixuture comprising cellobiohydrolase (CBH), β

-glucosidase, endoglucanase (EG), and EG with an inactivated cellulose binding domain enzyme, wherein said EG with an inactivated cellulose binding domain enzyme is present in said enzyme mixture in an amount of about 2 wt % to about 50 wt %, relative to the amount of said CBH and EG enzymes.

30. The method of claim 29, wherein said amount of EG with an inactivated cellulose binding domain enzyme in said enzyme mixture is from about 7 wt % to about 50 wt %.

31. The method of claim 30, wherein said amount of EG with an inactivated cellulose binding domain enzyme in said enzyme mixture is from about 7 wt % to about 30 wt %.