Covalently Immobilized Enzyme and Method To Make The Same

• US 20100078381A1
• Filed: 09/02/2009
• Published: 04/01/2010
• Est. Priority Date: 09/30/2008
• Status: Active Grant

• Current Assignee: Fresenius Medical Care Holdings Incorporated (Fresenius Medical Care AG)
• Original Assignee: Fresenius Medical Care Holdings Incorporated (Fresenius Medical Care AG)
• Inventors: Merchant, Stephen A.
• Granted Patent: US 8,497,107 B2
• Time in Patent Office: Days
• US Class Current: 210/632
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: B01D 2323/30 Cross-linking
• CPC Class Codes: B01D 67/0006 by chemical reactions in-situ polymerisation, polycondensation, cross-linking or reaction for manufacturing composite membranes B01D69/125
• CPC Class Codes: B01D 69/144 containing embedded or bound biomolecules
• CPC Class Codes: B01D 69/147 containing embedded adsorbents
• CPC Class Codes: B01D 71/021 Carbon
• CPC Class Codes: B01D 71/60 Polyamines
• CPC Class Codes: B01D 71/601 Polyethylenimine
• CPC Class Codes: B01J 20/267 Cross-linked polymers
• CPC Class Codes: B01J 2220/62 In a cartridge
• CPC Class Codes: C12N 11/06 attached to the carrier via a bridging agent
• CPC Class Codes: C12N 11/087 Acrylic polymers
• CPC Class Codes: C12N 11/089 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds

Abstract

A composition of enzyme, polymer, and crosslinker forms a network of covalently bound macromolecules. The covalently immobilized enzyme preparation has enzymatic activity, and retains stable activity when dried and stored at ambient conditions. Methods for preparing an immobilized enzyme and methods for using the enzyme are disclosed.

Claims

1. A method for preparing an immobilized enzyme, the method comprising:

forming an aqueous mixture of at least one polymer and at least one enzyme;

adding at least one crosslinker to the aqueous mixture to form a reaction mixture; and

maintaining the reaction mixture to immobilize the enzyme in a formation of covalently bound macromolecules.

2. The method of claim 1, wherein the polymer comprises a polyamine.

3. The method of claim 1, wherein the polymer comprises poly(ethylenimine), poly(propylenimine), poly(allylamine), poly(vinylpyridine), poly(vinylimidazole), poly(l-lysine), or any combination thereof.

4. The method of claim 1, wherein the polymer is branched poly(ethylenimine).

5. The method of claim 1, wherein the crosslinker comprises heterobifunctional polyethylene glycol, homobifunctional polyethylene glycol, or a combination thereof.

6. The method of claim 1, wherein the crosslinker comprises poly(ethylene glycol)diacrylate, tetra(ethylene glycol)diacrylate, poly(ethylene glycol)diglycidyl ether, ethylene glycol diglycidyl ether, dibromohexane, gluteraldehyde, epichlorohydrin, or any combination thereof.

7. The method of claim 1, wherein the crosslinker comprises one or more functional epoxy group, one or more functional acrylate group, one or more functional halide group, one or more functional carboxyl group, one or more functional aldehyde group, or a combination thereof.

8. The method of claim 1, wherein the crosslinker is ethylene glycol diglycidyl ether.

9. The method of claim 1, wherein the enzyme comprises glucose oxidase, lactate oxidase, peroxidase, creatinase, creatininase, sarcosine oxidase, glucose dehydrogenase, urease, or any combination thereof.

10. The method of claim 1, wherein the enzyme is urease.

11. The method of claim 1, wherein the enzyme is crude urease, raw urease or both.

12. The method of claim 1, wherein the enzyme is covalently bound to the crosslinker and to the polymer.

13. The method of claim 1, wherein the polymer is first dissolved in an aqueous solution, then the enzyme is added to the aqueous solution, and then the crosslinker is added to form the reaction mixture.

14. The method of claim 7, wherein a first functional group of the crosslinker reacts with the polymer, and a second functional group of the crosslinker reacts with the enzyme.

15. The method of claim 7, wherein a first functional epoxy group of the crosslinker reacts with an amine group of the polymer and a second functional epoxy group of the crosslinker reacts with an amine group of the enzyme.

16. The method of claim 15, wherein the amine group of the enzyme is from a lysine in the enzyme.

17. The method of claim 1, further comprising blending activated carbon into the mixture.

18. The method of claim 17, wherein the activated carbon comprises from about 1 percent to about 30 percent by weight of the formation.

19. The method of claim 1, further comprising blending zirconium phosphate into the mixture.

20. The method of claim 1, wherein the crosslinker is added in an amount to obtain a crosslink density of less than 40%.

21. The method of claim 1, wherein the crosslinker is added in an amount to obtain a crosslink density of from about 5% to about 15%.

22. A composition comprising a covalently immobilized enzyme prepared by the method of claim 1.

23. The composition of claim 22, wherein the enzyme is urease.

45. A sorbent cartridge comprising the composition of claim 23 as a layer in the sorbent cartridge.

46. The sorbent cartridge of claim 45, wherein said sorbent cartridge excludes the presence of alumina adjacent to said composition.

24. A dry, solid composition comprising a covalently immobilized enzyme and having enzymatic activity, the composition comprising a reaction product of (i) at least one enzyme, (ii) at least one polymer, and (iii) at least one crosslinker, in a formation of covalently bound macromolecules.

25. The composition of claim 24, wherein the polymer comprises poly(ethylenimine), poly(propylenimine), poly(allylamine), poly(vinylpyridine), poly(vinylimidazole), poly(l-lysine), or a combination thereof.

26. The composition of claim 24, wherein the polymer is branched poly(ethylenimine).

27. The composition of claim 24, wherein the crosslinker comprises heterobifunctional polyethylene glycol, homobifunctional polyethylene glycol, or a combination thereof.

28. The composition of claim 24, wherein the crosslinker comprises poly(ethylene glycol)diacrylate, tetra(ethylene glycol)diacrylate, poly(ethylene glycol)diglycidyl ether, ethylene glycol diglycidyl ether, dibromohexane, gluteraldehyde, epichlorohydrin, or a combination thereof.

29. The composition of claim 24, wherein the crosslinker comprises one or more functional epoxy groups, one or more functional acrylate groups, one or more functional halide groups, one or more functional carboxyl groups, one or more functional aldehyde groups, or a combination thereof.

30. The composition of claim 24, wherein the crosslinker is ethylene glycol diglycidyl ether.

31. The composition of claim 24, wherein the enzyme comprises glucose oxidase, lactate oxidase, peroxidase, creatinase, creatininase, sacrosine oxidase, glucose dehydrogenase, urease, or a combination thereof.

32. The composition of claim 24, wherein the enzyme is urease.

33. The composition of claim 24, wherein the enzyme is covalently bound to the crosslinker and to the polymer.

34. The composition of claim 24, wherein the enzyme retains at least about 10% of the enzymatic activity compared to non-immobilized enzyme.

35. The composition of claim 24, wherein the composition is a hydrogel.

36. The composition of claim 24, wherein the enzyme retains greater than 90% enzymatic activity when stored in a dry state, at ambient temperature and pressure, for at least 90 days, compared to enzymatic activity on day of preparation.

37. The composition of claim 24, further comprising activated carbon.

38. The composition of claim 37, wherein the activated carbon is present in an amount of from about 1% to about 30% by weight of the composition, the enzyme is present in an amount of from about 30% to about 55% by weight of the composition, the polymer is present in an amount of from about 25% to about 35% by weight of the composition, and the crosslinker is present in an amount to provide a crosslink density of no more than about 40%.

39. The composition of claim 24, further comprising zirconium phosphate.

41. A method for removing a compound from a system containing the compound, the method comprising treating the system with the immobilized enzyme of claim 24, and recovering a system substantially free of the compound.

42. The method of claim 41, wherein the compound comprises urea and the enzyme comprises urease.

43. The method of claim 41, wherein the system is a dialysis system.

44. The method of claim 41, further comprising treating the system with one or more ion-exchange material.

50. The composition of claim 24, wherein said polymer comprises a polyamine.

51. The composition of claim 24, wherein the composition has a crosslink density of less than 40%.

40. A composition comprising immobilized urease, at least one polymer, and at least one crosslinker, in a formation of covalently bound macromolecules.

47. A dry, solid composition comprising a covalently immobilized enzyme and having enzymatic activity, the composition comprising a reaction product of (i) at least one enzyme, (ii) at least one polymer, and (iii) at least one crosslinker, in a formation of covalently bound macromolecules, wherein the polymer comprises a polyamine, the crosslinker comprises heterobifunctional polyethylene glycol, homobifunctional polyethylene glycol, or a combination thereof, the enzyme is urease, and wherein said composition has a crosslink density of less than 40%, wherein the enzyme retains greater than 80% enzymatic activity when stored in a dry state, at ambient temperature and pressure, for at least 30 days, compared to enzymatic activity on day of preparation.

48. The composition of claim 47, wherein said urease is raw urease, crude urease or both.

49. The composition of claim 47, wherein said urease is a purified urease.