Stabilization of Enzymes

• US 20080213858A1
• Filed: 01/27/2005
• Published: 09/04/2008
• Est. Priority Date: 01/28/2004
• Status: Active Grant

• Current Assignee: CSIR
• Original Assignee: CSIR
• Inventors: Brady, Dean, Rolfes, Heidi, Moolman, Francis Sean, Jordaan, Justin, Chetty, Avashnee Shamparkesh
• Granted Patent: US 7,700,335 B2
• Time in Patent Office: Days
• US Class Current: 435/177
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Abstract

A process for producing enzymes structures includes providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase. The one liquid phase is a hydrophilic phase, while the other liquid phase is a hydrophobic phase which is immiscible with the hydrophilic phase. Enzyme molecules are located at or within interfacial boundaries of the droplets and the second liquid phase. The enzyme molecules of the respective droplets are cross-linked so that individual enzyme structures, which are stable and in which the enzymes are immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets.

Claims

1. A process for producing enzyme particles, which process comprising includes

providing an emulsion of droplets of a first liquid phase dispersed in a second liquid phase, with the one liquid phase being a hydrophilic phase and the other liquid phase being a hydrophobic phase which is immiscible with the hydrophilic phase, and with enzyme molecules being located at or within interfacial boundaries of the droplets and the second liquid phase;

cross-linking the enzyme molecules of the respective droplets so that individual enzyme particles, which are stable and in which the enzymes are immobilized with a majority of active sites of the enzymes being orientated either internally or externally, are formed from individual droplets; and

recovering the individual enzyme particles from the second liquid phase.

2. The process according to claim 1, wherein the individual particles have openings so that the liquid phases can pass in or out of the particles.

3. The process according to claim 1, wherein individual particles are liquid impervious.

4. The process according to claim 1, further comprising adding to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion, a modifier for modifying the hydrophobicity and/or charge of the enzyme.

5. The process according to claim 1, wherein the enzyme is a lipase.

6. The process according to claim 5, wherein the lipase is selected from the group consisting of Pseudomonas cepacia lipase, Pseudomonas fluorescens lipase, Pseudomonas alcaligenes lipase, Candida rugosa lipase, Candida antarctica lipase A, Candida antarctica lipase B, Candida utilis lipase, Thermomyces lanuginosus lipase, Rhizomucor miehei lipase, Aspergillus niger lipase, Aspergillus oryzae lipase, Penicillium sp lipase, Mucor javanicus lipase, Mucor miehei lipase, Rhizopus arrhizus lipase, Rhizopus delemer lipase, Rhizopus japonicus lipase, Rhizopus niveus lipase, and Porcine Pancreatic lipase.

7. The process according to claim 5, wherein the provision of the emulsion is effected by dissolving the enzyme in the hydrophilic or W phase and forming the emulsion by mixing the enzyme containing hydrophilic phase with the hydrophobic or O phase.

8. The process according to claim 7, further comprising selectively precipitating the enzyme at the interface when the emulsion is a O/W emulsion in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, or within the droplet volume, when the emulsion is a W/O emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase.

9. The process according to claim 7, wherein the cross-linking of the enzyme molecules is effected by means of a cross-linking agent which is added to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion.

10. The process according to claim 9, further comprising adding to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion, a temporary protectant that occupies active sites of the enzyme during the cross-linking, thereby inhibiting occupation of or reaction with the active sites by the cross-linking agent.

11. The process according to claim 7, further comprising adding an amino acid to the emulsion to inhibit agglomeration of the individual enzyme particles.

12. The process according to claim 7, further comprising recovering the enzyme particles from the second liquid phase.

13. The process according to claim 7, further comprising extracting the first liquid phase from the enzyme particles.

14. The process according to claim 7, wherein the hydrophilic phase comprises water.

15. The process according to claim 7, wherein the hydrophilic phase comprises a polyethylene glycol.

16. The process according claim 7, wherein the hydrophobic phase comprises an oil;

a hydrocarbon;

an ether;

or an ester.

17. The process according claim 7, wherein the emulsion is a W/O emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase, with a second enzyme, co factor and/or mediator being present in the hydrophilic phase.

18. The process according to claim 5, wherein a triglyceride, which is hydrolysable by lipase, is used as the hydrophobic phase, with an O/W emulsion, in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, being formed and with the dispersed hydrophobic phase contained within the cross-linked particles being hydrolyzed by the lipase during and after the cross-linking reaction.

19. The process according claim 7, wherein an initial O/W emulsion, in which hydrophobic phase droplets are dispersed in a continuous hydrophilic phase, is formed, with the process including, before effecting the cross-linking, centrifuging the emulsion and separating a concentrated emulsion from a dilute hydrophilic phase, to increase lipase purity;

and inverting the emulsion to form a W/O emulsion in which hydrophilic phase droplets are dispersed in a continuous hydrophobic phase, by the addition of a surfactant with a lower HLB value.

20. The process according claim 1, wherein, to impart specific properties to the enzyme particles, a modifier is added to the hydrophilic phase and/or to the hydrophobic phase and/or to the emulsion.

21. The process according to claim 20, wherein the modifier is a surfactant, for imparting enhanced enzyme activity and improved emulsion stability.

22. The process according to claim 20, wherein the modifier is a precipitator for precipitating the enzyme onto the emulsion interfaces.

23. The process according to claim 20, wherein the modifier is an additive for modifying the pH;

ionic strength;

viscosity;

magnetic properties;

agglomeration tendency; and

/or zeta potential of the emulsion and/or the enzyme particles.

30. The process according to claim 14, wherein the hydrophilic phase further comprises a buffer in the water.

31. The process according to claim 15, wherein the hydrophilic phase further comprises water admixed with the polyethylene glycol.

24. An enzyme particle, which comprises cross-linked enzyme molecules so that the particle is stable, with the particle being hollow, and in which the enzymes are immobilized, with a majority of active sites of the enzymes being orientated either internally or externally.

25. The enzyme particle according to claim 24, which is spherical.

26. The enzyme particle according to claim 24, which contains, in its lumen, a liquid.

27. An The enzyme particle according to claim 24, wherein the enzyme is a lipase.

28. The enzyme particle according to claim 27, wherein the lipase is selected from the group consisting of Pseudomonas cepacia lipase, Pseudomonas fluorescens lipase, Pseudomonas alcaligenes lipase, Candida rugosa lipase, Candida antarctica lipase A, Candida antarctica lipase B, Candida utilis lipase, Thermomyces lanuginosus lipase, Rhizomucor miehei lipase, Aspergillus niger lipase, Aspergillus oryzae lipase, Penicillium sp lipase, Mucor javanicus lipase, Mucor miehei lipase, Rhizopus arrhizus lipase, Rhizopus delemer lipase, Rhizopus japonicus lipase, Rhizopus niveus lipase, and Porcine Pancreatic lipase.

29. A method of carrying out a reaction, comprising allowing a reaction medium to undergo a reaction in the presence of a plurality of the enzyme particles according to claim 24, with the reaction thus being catalyzed by the enzyme particles.