Claims
- 1. A method of purification of an enzyme comprising:
a) flocculating a fermentation broth containing bacterial cells containing the desired enzyme with a flocculating agent under agitation conditions, wherein a resultant mixture is formed; b) washing the resultant mixture with a buffered medium; c) releasing the enzyme contained in the cells of the resultant mixture; d) extracting by centrifugation a centrate solution containing enzymes; and e) filtering the centrate solution, wherein a resultant permeate is formed; wherein the resultant permeate contains the purified enzyme.
- 2. The method of claim 1, further comprising:
f) concentrating the resultant permeate.
- 3. The method of claim 2, further comprising:
g) neutralizing the resultant permeate.
- 4. The method of claim 1, wherein the enzyme is derived from a mixed population of organisms.
- 5. The method of claim 4, wherein the enzyme is derived from an isolate of a mixed population of organisms.
- 6. The method of claim 1, wherein the enzyme is a thermophilic enzyme.
- 7. The method of claim 1, wherein the enzyme is an amylase.
- 8. The method of claim 7, wherein the amylase is an alpha-amylase.
- 9. The method of claim 1, wherein the enzyme is a cellulase.
- 10. The method of claim 1, wherein the enzyme is a catalase, α-galactosidase, amidase, endoglucanase, glycosidase, esterase, phosphatase, transaminase, aminotransferase, nitrilase, lipase, protease, laccase, α-glucosidase, glucoamylase, epoxide, hydrolase, xylanase, polymerase, phospholipase C, phytase, nitroreductase or hydrolase.
- 11. The method of claim 1, wherein the flocculating agent is cationic.
- 12. The method of claim 11, wherein the flocculating agent is E-4244.
- 13. The method of claim 1, wherein the flocculating agent is anionic.
- 14. The method of claim 1, wherein the flocculating agent is non-ionic.
- 15. The method of claim 1, wherein the releasing of the enzyme is by adjusting the pH of the resultant mixture to a solubilizing pH.
- 16. The method of claim 15, wherein the solubilizing pH is an alkaline pH.
- 17. The method of claim 16, wherein the alkaline pH is in the range of about 10 to 11.5.
- 18. The method of claim 17, wherein the alkaline pH is in the range of about 10.8 to 11.1.
- 19. The method of claim 1, wherein the releasing of the enzyme is by heat treatment comprising raising the temperature to about 70° C. for about 30 minutes.
- 20. The method of claim 1 further comprising increasing enzyme solubility in the resultant mixture, by a method selected from adjusting the pH, adjusting the temperature, performing a buffer exchange, including additional additives in the resultant mixture, or adding detergents to the resultant mixture.
- 21. A method of purification of an enzyme comprising:
a) subjecting a fermentation broth to a heat-killing procedure, wherein a resultant heat-killed broth is formed; b) washing the heat-killed broth with a buffered medium; c) releasing the enzyme contained in the cells of a fermentation broth; and d) extracting the enzyme by microfiltration, wherein a resultant solution is formed; wherein the resultant solution contains the purified enzyme.
- 22. The method of claim 21, further comprising:
e) concentrating the resultant solution.
- 23. The method of claim 22, further comprising:
f) neutralizing the resultant solution.
- 24. The method of claim 21, wherein the enzyme is derived from a mixed population of organisms.
- 25. The method of claim 21, wherein the enzyme is derived from an isolate of a mixed population of organisms.
- 26. The method of claim 21, wherein the enzyme is a thermophilic enzyme.
- 27. The method of claim 21, wherein the enzyme is an amylase.
- 28. The method of claim 27, wherein the amylase is an alpha-amylase.
- 29. The method of claim 21, wherein the enzyme is a cellulase.
- 30. The method of claim 21, wherein the enzyme is a catalase, α-galactosidase, amidase, endoglucanase, glycosidase, esterase, phosphatase, transaminase, aminotransferase, nitrilase, lipase, protease, laccase, α-glucosidase, glucoamylase, epoxide, hydrolase, xylanase, polymerase, phospholipase C, phytase, nitroreductase or hydrolase.
- 31. The method of claim 21, wherein the releasing of the enzyme is by adjusting the pH of the resultant mixture to a solubilizing pH.
- 32. The method of claim 31, wherein the solubilizing pH is an alkaline pH.
- 33. The method of claim 32, wherein the alkaline pH is in the range of about 10 to 11.5.
- 34. The method of claim 33, wherein the alkaline pH is in the range of about 10.8 to 11.1.
- 35. The method of claim 21, wherein the releasing of the enzyme is by heat treatment comprising raising the temperature to about 70° C. for about 30 minutes.
- 36. The method of claim 21 further comprising increasing enzyme solubility in the resultant mixture, by a method selected from adjusting the pH, adjusting the temperature, performing a buffer exchange, including additional additives in the resultant mixture, or adding detergents to the resultant mixture.
- 37. A method of purification of an alpha-amylase comprising:
a) subjecting a fermentation broth containing bacterial cells containing alpha-amylase to a heat-killing procedure, wherein a resultant heat-killed broth is formed; b) flocculating the resultant heat-killed broth with a flocculating agent under agitation conditions, wherein a resultant mixture is formed; c) washing the resultant mixture with a buffered medium; d) releasing the enzyme contained in the cells of the resultant mixture by adjusting the pH to the range of 10-11.5; e) extracting by centrifugation a centrate solution containing enzymes; and f) filtering the centrate solution, wherein a resultant permeate is formed; wherein the resultant permeate contains the purified alpha-amylase.
- 38. The method of claim 37, further comprising:
g) concentrating the resultant permeate.
- 39. The method of claim 38, further comprising:
h) neutralizing the resultant permeate.
- 40. A method of purification of a cellulase comprising:
a) flocculating a fermentation broth containing bacterial cells containing cellulase with a flocculating agent under agitation conditions, wherein a resultant mixture is formed; b) subjecting the resultant mixture to a heat-killing procedure, wherein a resultant heat-killed broth is formed; c) extracting by centrifugation a centrate solution containing enzymes; and d) filtering the centrate solution, wherein a resultant permeate is formed; wherein the resultant permeate contains the purified cellulase.
- 41. The method of claim 40, further comprising:
e) concentrating the resultant permeate.
RELATED APPLICATION DATA
[0001] This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application Serial No. 60/291,122, filed May 14, 2001, which is hereby incorporated by reference in its entirety.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60291122 |
May 2001 |
US |