Enzyme preparation having affinity for water-immiscible liquids

Claim 2

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.

Claim 3

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

Claim 4

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

Claim 5

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

Claim 6

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

Claim 7

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

Claim 8

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

Claim 9

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.

Claim 10

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

Claim 11

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.

Claim 12

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

Claim 13

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

Claim 14

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.

Claim 15

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

Claim 16

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.

Claim 17

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.

Claim 18

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.

Claim 19

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.

Claim 20

A process according to claim 16 wherein said polymeric support comprises pendant anhydride groups modified by the reaction with an amine of formula R.NH 2 or R 2 NH wherein R is non-polar hydrocarbon in an aprotic non-aqueous polar solvent.

Claim 21

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

Claim 22

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

Claim 23

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

Claim 24

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.

Claim 25

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.

Claim 26

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.

Claim 27

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

Claim 28

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.

Claim 29

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

Claim 30

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

Claim 31

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.

Claim 32

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.