Crosslinked protein crystal formulations and their use as catalysts in organic solvents

• US 5,932,212 A
• Filed: 05/24/1996
• Issued: 08/03/1999
• Est. Priority Date: 05/24/1996
• Status: Expired due to Term

• Current Assignee: Ajinomoto Althea, Inc. (Ajinomoto Co., Inc.)
• Original Assignee: Altus Biologics, Inc.
• Inventors: Khalaf, Nazer K.
• Primary Examiner(s): Achutamurthy, Ponnathapura
• Assistant Examiner(s): WESSENDORF, TERESA D
• Application Number:
  US08/652,964
• Time in Patent Office: 1,166 Days
• Field of Search: 424/94.6, 424/94.5, 424/94.3, 424/94.2, 514/4, 514/2, 530/287.1, 530/350, 435/183, 435/188, 435/188.5
• US Class Current: 424/94.6
• CPC Class Codes: C12N 11/00 Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase

Abstract

The present invention relates to the application of biocatalysis technology for performing selective chemical reactions. In one embodiment, this invention relates to crosslinked protein crystal formulations and their use as catalysts in chemical reactions involving organic solvents. This invention also provides methods for producing crosslinked protein crystal formulations and methods using them to optimize chemical reactions in organic solvents, including those used in industrial scale chemical processes.

Claims

1. A crosslinked enzyme crystal formulation, said formulation comprising:

(a) a crosslinked enzyme crystal; and

(b) a surfactant, wherein said surfactant is selected from the group consisting of anionic surfactants, cationic surfactants and non-ionic surfactants;

said formulation having an activity in an organic solvent or an aqueous-organic solvent mixture which is at least 1.7 times greater than the activity of the equivalent amount of said enzyeme in either crude form or pure form.

2. The crosslinked enzyme crystal formulation according to claim 1, said formulation having activity in an organic solvent or an aqueous-organic solvent mixture which is between about 1.7 times and about 90 times greater than the activity of the equivalent amount of said enzyme in either crude form or pure form.

11. The crosslinked enzyme crystal formulation according to claim 1, wherein said surfactant comprises between about 10% and about 70% by weight of said formulation.

12. The crosslinked enzyme crystal formulation according to claim 11, wherein said surfactant comprises between about 25% and about 45% by weight of said formulation.

13. The crosslinked enzyme crystal formulation according to any one of claims 1, 3 or 9, wherein said anionic surfactant is selected from the group consisting of linear alkylbenzene sulphonate, alpha-olefin sulphonate, alkyl sulphate, alcohol ethoxy sulfate, carboxylic acids, sulfuric esters and alkane sulfonic acids.

14. The crosslinked enzyme crystal formulation according to any one of claims 1, 3 or 9, wherein said cationic surfactant is selected from the group consisting of amines, amine salts, sulfonium, phosphonium and quarternary ammonium compounds.

15. The crosslinked enzyme crystal formulation according to any one of claims 1, 3 or 9, wherein said non-ionic surfactant is selected from the group consisting of nonyl phenol ethoxylate, alcohol ethoxylate, sorbitan trioleate, non-ionic block copolymer surfactants, polyethylene oxide, polyethylene oxide substituted phenolic alcohols and polyethylene oxide substituted fatty acids.

16. The crosslinked enzyme crystal formulation according to any one of claims 1, 3 or 9, wherein said enzyme crystal is a microcrystal.

17. The crosslinked enzyme crystal formulation according to any one of claims 1, 3 or 9, wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, transferases and oxidoreductases.

18. The crosslinked enzyme crystal formulation according to claim 17, wherein said enzyme is a hydrolase.

19. The crosslinked enzyme crystal formulation according to claim 18, wherein said hydrolase is selected from the group consisting of thermolysin, elastase, esterase, lipase, nitrilase, hydantoinase, protease, asparaginase, urease and lysozyme.

20. The crosslinked enzyme crystal formulation according to any one of claims 1, 3 or 9, wherein said formulation is in lyophilized form.

21. The crosslinked enzyme crystal formulation according to any one of claims 1, 3 or 9, wherein said organic solvent comprises an organic solvent selected from the group consisting of octanes, diols, polyols, polyethers and water soluble polymers.

22. The crosslinked enzyme crystal formulation according to claim 21, wherein said organic solvent is selected from the group consisting of toluene, octane, tetrahydrofuran, acetone, pyridine, diethylene glycol, 2-methyl-2,4-pentanediol, poly(ethylene glycol), triethylene glycol, 1,4-butanediol, 1,2-butanediol, 2,3,-dimethyl-2,3-butanediol, 1,2-butanediol, dimethyl tartrate, monoalkyl ethers of poly(ethylene glycol), dialkyl ethers of poly(ethylene glycol), and polyvinylpyrrolidone.

39. A reactive topical composition comprising a catalytically effective amount of a crosslinked enzyme crystal formulation according to any one of claims 1, 3 or 9 and a pharmaceutically acceptable carrier.

40. The reactive topical composition according to claim 39, wherein said crosslinked enzyme crystal formulation comprises between about 0.1% and about 99% by weight of said composition.

41. The reactive topical composition according to claim 40, wherein said crosslinked enzyme crystal formulation comprises between about 1% and about 10% by weight of said composition.

3. A crosslinked enzyme crystal formulation, said formulation comprising:

(a) a crosslinked enzyme crystal; and

(b) a surfactant, wherein said surfactant is selected from the group consisting of anionic surfactants, cationic surfactants and non-ionic surfactants;

said formulation having a specific activity per milligram of solid in an organic solvent or an aqueous-organic solvent mixture which is at least 4.3 times greater than that of said enzyme in either crude form or pure form.

4. The crosslinked enzyme crystal formulation according to claim 3, said formulation having a specific activity per milligram of solid in an organic solvent or an aqueous-organic solvent mixture which is between about 4 times and about 442 times greater than that of said enzyme in either crude form or pure form.

5. The crosslinked enzyme crystal formulation according to claim 3, said formulation having a specific activity per milligram of solid in an organic solvent or an aqueous-organic solvent mixture which is at least 50 times greater than that of said enzyme in either crude form or pure form.

6. The crosslinked enzyme crystal formulation according to claim 3, said formulation having a specific activity per milligram of solid in an organic solvent or an aqueous-organic solvent mixture which is at least 100 times greater than that of said enzyme in either crude form or pure form.

7. The crosslinked enzyme crystal formulation according to claim 3, said formulation having a specific activity per milligram of solid in an organic solvent or an aqueous-organic solvent mixture which is at least 200 times greater than that of said enzyme in either crude form or pure form.

8. The crosslinked enzyme crystal formulation according to claim 3, said formulation having a specific activity per milligram of solid in an organic solvent or an aqueous-organic solvent mixture which is at least 300 times greater than that of said enzyme in either crude form or pure form.

9. A crosslinked enzyme crystal formulation, said formulation comprising:

(a) a crosslinked enzyme crystal; and

(b) a surfactant, wherein said surfactant is selected from the group consisting of anionic surfactants, cationic surfactants and non-ionic surfactants;

said formulation having an activity in an organic solvent or an aqueous-organic solvent mixture which is at least 19 times greater than the activity of crosslinked enzyme crystals containing no surfactant.

10. The crosslinked enzyme crystal formulation according to claim 9, said formulation having activity in an organic solvent or an aqueous-organic solvent mixture which is between about 19 times and about 100 times greater than the activity of crosslinked enzyme crystals containing no surfactant.

23. A method for increasing the activity of crosslinked enzyme crystals in an organic solvent or a mixed aqueous-organic solvent mixture, said method comprising the steps of:

(a) combining the crosslinked enzyme crystals with a surfactant to produce a combination; and

(b) drying the combination of crosslinked enzyme crystals and surfactant in the presence of an organic solvent to form a crosslinked enzyme crystal formulation;

said formulation having an activity in an organic solvent or an aqueous-organic solvent mixture which is at least 1.7 times greater than the activity of the equivalent amount of said enzyme in either crude form or pure form.

24. The method according to claim 23, wherein, in step (a), the combination comprises a weight ratio of crosslinked enzyme crystals to surfactant between about 1:

1 and about 1;

5.

25. The method according to claim 24, wherein, in step (a), the combination comprises a weight ratio of crosslinked enzyme crystals to surfactant between about 1:

1 and about 1;

2.

26. The method according to claim 23, wherein, in step (a), the crosslinked enzyme crystals and the surfactant are combined for a period of time between about 5 minutes and about 24 hours.

27. The method according to claim 26, wherein, in step (a), the crosslinked enzyme crystals and the surfactant are combined for a period of time between about 30 minutes and about 24 hours.

28. The method according to claim 23, wherein the surfactant comprises between about 10% and about 70% by weight of the crosslinked enzyme crystal formulation produced in step (b).

29. The method according to claim 28, wherein the surfactant comprises between about 25% and about 45% by weight of the crosslinked enzyme crystal formulation produced in step (b).

30. The method according to claim 23, wherein said surfactant is selected from the group consisting of anionic surfactants, cationic surfactants and non-ionic surfactants.

31. The method according to claim 30, wherein said anionic surfactant is selected from the group consisting of linear alkylbenzene sulphonate, alpha-olefin sulphonate, alkyl sulphate, alcohol ethoxy sulfate, carboxylic acids, sulfuric esters and alkane sulfonic acids.

32. The method according to claim 30, wherein said cationic surfactant is selected from the group consisting of amines, amine salts, sulfonium, phosphonium and quarternary ammonium compounds.

33. The method according to claim 30, wherein said non-ionic surfactant is selected from the group consisting of nonyl phenol ethoxylate, alcohol ethoxylate, sorbitan trioleate, non-ionic block copolymer surfactants, polyethylene oxide, polyethylene oxide substituted phenolic alcohols and polyethylene oxide substituted fatty acids.

34. The method according to claim 30, wherein said enzyme is selected from the group consisting of hydrolases, isomerases, lyases, ligases, transferases and oxidoreductases.

35. The method according to claim 34, wherein said enzyme is a hydrolase.

36. The method according to claim 35, wherein said hydrolase is selected from the group consisting of thermolysin, elastase, esterase, lipase, asparaginase, nitrilase, hydantoinase, protease, urease and lysozyme.

37. The method according to claim 30, wherein said organic solvent comprises an organic solvent selected from the group consisting of octanes, diols, polyols, polyethers, and water soluble polymers.

38. The method according to claim 30, wherein said organic solvent is selected from the group consisting of toluene, octane, tetrahydrofuran, acetone, pyridine, diethylene glycol, 2-methyl-2,4-pentanediol, poly(ethylene glycol), triethylene glycol, 1,4-butanediol, 1,2-butanediol, 2,3,-dimethyl-2,3-butanediol, 1,2-butanediol, dimethyl tartrate, monoalkyl ethers of poly(ethylene glycol), dialkyl ethers of poly(ethylene glycol) and polyvinylpyrrolidone.