Enzyme preparation having affinity for water-immiscible liquids

• US 4,267,273 A
• Filed: 04/12/1979
• Issued: 05/12/1981
• Est. Priority Date: 08/13/1974
• Status: Expired due to Term

• Current Assignee: Beecham Group PLC (GlaxoSmithKline PLC)
• Original Assignee: Beecham Group PLC (GlaxoSmithKline PLC)
• Inventors: Smith, Richard A. G.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US06/029,204
• Time in Patent Office: 761 Days
• Field of Search: 435/44, 435/45, 435/174, 435/177, 435/178, 435/179, 435/180, 435/181, 435/231, 435/814
• US Class Current: 435/44
• CPC Class Codes: B01J 31/003 containing enzymes
• CPC Class Codes: B01J 31/06 containing polymers organometallic polymers B01J31/123; polymer-bound organometallic complexes B01J31/165; coordination polymers B01J31/1691
• CPC Class Codes: B01J 31/061 Chiral polymers
• CPC Class Codes: C12M 21/18 Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
• CPC Class Codes: C12M 27/00 Means for mixing, agitating or circulating fluids in the vessel by introduction of gas C12M29/06, C12M29/14, mixing in general or mixers per se B01F; mixing in apparatus for chemical or physical processes B01J
• CPC Class Codes: C12M 29/18 External loop; Means for reintroduction of fermented biomass or liquid percolate loop type reactors for chemical or physical processes B01J19/2435
• CPC Class Codes: C12M 47/10 Separation or concentration of fermentation products bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02
• CPC Class Codes: C12N 11/082 obtained by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: C12N 11/089 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: C12N 11/096 Polyesters; Polyamides
• CPC Class Codes: C12N 11/10 the carrier being a carbohydrate
• CPC Class Codes: C12P 1/00 Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• CPC Class Codes: Y10S 435/814 Enzyme separation or purification

Abstract

An enzyme preparation is prepared having terminal non-polar groups incorporated therein so that the enzyme preparation has affinity for a water-immiscible liquid. The enzyme preparation can be separated from an aqueous reaction mixture by contacting the mixture with the water-immiscible liquid, permitting the enzyme preparation to become associated with the water-immiscible liquid and separating the water-immiscible liquid containing the associated enzyme from the reaction mixture.

Claims

1. In an enzyme preparation having an enzyme portion and a polymeric support in which the enzyme portion is covalently bound directly to the polymeric support or is covalently bound to an inert bridging group which bridging group is in turn covalently bound to said polymeric support, the improvement which comprises the presence of a plurality of hydrophobic groups covalently bound to either or both of said enzyme portion and said polymeric support, said hydrophobic groups being hydrophobic at least by reason of a free terminal non-polar hydrocarbon portion of six or more carbon atoms, and being present in said enzyme preparation in such proportion as to impart, without deactivation of the enzyme portion, sufficient overall hydrophobic properties to said enzyme preparation to permit separation of the preparation from aqueous media through preferential association with an inert water-immiscible organic liquid.

2. An enzyme preparation according to claim 1 wherein the polymeric support is a polysaccharide material.

3. An enzyme preparation according to claim 2 wherein the polymeric support is dextran or sepharose.

4. An enzyme preparation according to claim 1 wherein the polymeric support is a copolymer of an oligosacccharide and epichlorhydrin.

5. An enzyme preparation according to claim 4 wherein the polymeric support is a copolymer of sucrose and epichlorhydrin.

6. An enzyme preparation according to claim 1 wherein the polymeric support comprises anhydride groups on the polymer backbone.

7. An enzyme preparation according to claim 6 wherein the polymeric support is a copolymer of methyl vinyl ether and maleic anhydride.

8. An enzyme preparation according to claim 1 wherein the enzyme of the enzyme portion is amylase, asparaginase neutral protease, alkaline protease, chymotrypsin, cellulase, dextranase, lipase, oxynitrilase, pepsin, penicillin acylase or trypsin.

9. An enzyme preparation according to claim 8 wherein the enzyme is penicillin acylase.

10. An enzyme preparation according to claim 1 wherein the non-polar groups are selected from the group consisting of alkyl of 6 to 30 carbon atoms, alkenyl of 6 to 50 carbon atoms, cycloalkyl of 6 to 20 carbon atoms, phenyl, unsubstituted or substituted with one to three alkyl groups of 1 to 3 carbon atoms, benzyl 2-phenethyl, 4-phenylbutyl and 2-tolyethyl.

11. An enzyme preparation according to claim 10 wherein the non-polar group is alkyl of 6 to 30 carbon atoms.

12. An enzyme preparation according to claim 11 wherein the non-polar group is n-decyl, n-dodecyl or n-octadecyl.

13. An enzyme preparation according to claim 1 wherein said inert bridging group is derived from an aliphatic α

,ω

-diamine of 2 to 10 carbon atoms, a water soluble dialdehyde or said diamine and said aldehyde.

14. An enzyme preparation according to claim 1 wherein the enzyme is linked directly to the polymeric support.

15. An enzyme preparation consisting essentially of an enzyme covalently bound directly to a plurality of hydrophobic groups, said hydrophobic groups being hydrophobic at least by reason of a free terminal non-polar hydrocarbon portion of six or more carbon atoms, and being present in said enzyme preparation in such proportion as to impart, without deactivation of the enzyme, sufficient overall hydrophobic properties to said enzyme preparation to permit separation of the preparation from aqueous media through preferential association with an inert water-immiscible organic liquid.

16. The process for the preparation of an enzyme preparation having an enzyme portion and a polymeric support in which the enzyme portion is covalently bound directly to the polymeric support or is covalently bound to an inert bridging group which bridging group is in turn covalently bound to said polymeric support said preparation further having a plurality of hydrophobic groups covalently bound to said polymeric support, said hydrophobic groups being hydrophobic at least by reason of a free terminal non-polar hydrocarbon portion of six or more carbon atoms, and being present in said enzyme preparation in such proportion as to impart, without deactivation of the enzyme portion, sufficient overall hydrophobic properties to said enzyme preparation to permit separation of the preparation from aqueous media through preferential association with an inert water-immiscible organic liquid, which process comprises chemically joining an enzyme or an activated derivative thereof (a) with a polymeric support having pendant functional groups capable of covalently reacting with said enzyme or activated derivative thereof and to which support has also been covalently bound said terminal hydrophobic groups or (b) with a polymeric support to which both said terminal hydrophobic groups and said bridging groups have been covalently bound, said bridging groups having terminal functional groups capable of covalently reacting with said enzyme or activated derivative thereof.

17. A process according to claim 16 wherein said polymeric support comprises pendant anhydride groups modified by the reaction with an amine of formula R.NH₂ or R₂ NH wherein R is non-polar hydrocarbon in an aprotic non-aqueous polar solvent.

18. A process according to claim 17 wherein the non-aqueous aprotic solvent is N,N-dimethylformamide.

19. A process according to claim 17 which is carried out in aqueous solution or suspension.

20. A process according to claim 19 which is carried out at a pH in the range pH 4 to pH 9.

21. In an enzymic reaction wherein an enzyme preparation is contacted, in aqueous medium, with a substrate for said enzyme, the enzyme preparation is separated from the reaction mixture and the reaction product is recovered, the improvement permitting re-use of the separated enzyme preparation which comprises utilizing an enzyme preparation having an enzyme portion, a polymeric support said enzyme portion being covalently bound directly to the polymeric support or covalently bound to an inert bridging group which bridging group is in turn covalently bound to said polymeric support, and a plurality of hydrophobic groups covalently bound to either or both of said enzyme portion and said polymeric support, said hydrophobic groups being hydrophobic at least by reason of a free terminal non-polar hydrocarbon portion of six or more carbon atoms, and being present in said enzyme preparation in such proportion as to impart, without deactivation of the enzyme portion, sufficient overall hydrophobic properties to said enzyme preparation to permit separation of the preparation from aqueous media through preferential association with an inert water-immiscible organic liquid.

22. A process according to claim 21 wherein the inert water-immiscible organic liquid is an alkane, an aromatic hydrocarbon, a higher aliphatic ester, or an aliphatic alcohol of 2 to 14 carbon atoms.

23. A process according to claim 21 wherein contact between said enzyme preparation and said inert water-immiscible liquid is established by agitation.

24. A process according to claim 23 wherein the contact between said enzyme preparation and said water-immiscible liquid is established prior to said enzymic reaction.

25. A process according to claim 21 wherein said enzyme is penicillin acylase.

26. A process according to claim 25 wherein the substrate is benzylpenicillin or phenoxymethylpenicillin.

27. A process for the preparation of 6-amino-penicillanic acid from benzylpenicillin, which process comprises contacting benzylpenicillin in aqueous medium with an inert water-immiscible organic liquid and an enzyme preparation comprising penicillin acylase covalently attached to a plurality of free terminal non-polar groups in such proportion as to impart to the preparation sufficient overall hydrophobic properties to cause preferential association with said inert water-immiscible organic liquid, thereafter allowing the aqueous and water-immiscible layers to separate, separating said water-immiscible layer and the associated enzyme preparation from the aqueous reaction mixture, and recovering the 6-aminopenicillanic acid from the aqueous layer.