Process for the purification of glucuronoglycosaminoglycan hyaluronate lyase

Claim 2

A process for the preparation of very pure and highly active glucuronoglycosaminoglycan hyaluronate lyase, which comprises contacting a crude lyase-containing solution with a cross-linked polyacrylic divinyl-benzene resin containing carboxyl groups as active groups, eluting the absorbed lyase from the resin, dialyzing the eluate fractions which are rich in the desired lyase against a saturated aqueous solution of ammonium sulphate and further purifying the enzyme concentrate thus obtained by contacting said concentrate with a cross-linked dextran gel containing active carboxymethyl groups, followed by elution of the gel to give a solution of the desired lyase.

Claim 3

A process according to claim 1, wherein the cross-linked polyacrylic divinyl-benzene resin is equilibrated at ambient temperature with a 0.1M phosphate buffer (pH 6.0).

Claim 4

A process according to claim 1, wherein the crude enzyme is applied to the cross-linked polyacrylic divinyl-benzene resin in a 0.1M phosphate buffer (pH 6.0).

Claim 5

A process according to claim 1, wherein the cross-linked polyacrylic divinyl-benzene resin is washed with a 0.1M phosphate buffer (pH 6.0) and the lyase is then eluted with a 0.3M phosphate buffer (pH 7.7).

Claim 6

A process according to claim 1, wherein the dialysis is carried out at a temperature of about 4° C..

Claim 7

A process according to claim 1, wherein the dialysis concentrate is dissolved in 0.1M acetate buffer (pH 5.0), applied at about 4° C. to a cross-linked dextran gel containing active carboxymethyl groups which has been equilibrated with 0.1M acetate buffer (pH 5.0) and the gel then eluted with 1.0M acetate buffer (pH 5.0).

Claim 8

A process for the preparation of very pure and highly active glucuronoglycosaminoglycan hyaluronate lyase, which comprises contacting a crude lyase-containing solution with a cross-linked polyacrylic divinyl-benzene resin containing carboxyl groups as active groups, eluting the adsorbed lyase from the resin, dialyzing the eluate fractions which are rich in the desired lyase against a saturated aqueous solution of ammonium sulphate and further purifying the enzyme concentrate thus obtained by containing said concentrate with a cross-linked dextran gel which does not contain active carboxymethyl groups, followed by elution of the gel to give a solution of the desired lyase.

Claim 9

A process for the preparation of very pure and highly active glucuronoglycosaminoglycan hyaluronate lyase, which comprises contacting a crude lyase-containing solution with a cross-linked polyacrylic divinyl-benzene resin containing carboxyl groups as active groups, eluting the adsorbed lyase from the resin, dialyzing the eluate fractions which are rich in the desired lyase against a saturated aqueous solution of ammonium sulphate and further purifying the enzyme concentrate thus obtained by containing said concentrate with a cross-linked dextran gel which does not contain active carboxymethyl groups, followed by elution of the gel to give a solution of the desired lyase.

Claim 10

A process according to claim 7, wherein the eluate obtained from the cross-linked dextran gel which does not contain active carboxymethyl groups is further contacted with a cross-linked dextran gel containing active carboxymethyl groups, followed by elution of the gel to give a solution of the desired lyase.

Claim 11

A process according to claim 7, wherein the cross-linked polyacrylic divinyl-benzene resin is equilibrated at ambient temperature with a 0.1M phosphate buffer (pH 6.0).

Claim 12

A process according to claim 7, wherein the crude enzyme is applied to the cross-linked polyacrylic divinyl-benzene resin in a 0.1M phosphate buffer (pH 6.0).

Claim 13

A process according to claim 7, wherein the cross-linked polyacrylic divinyl-benzene resin is washed with a 0.1M phosphate buffer (pH 6.0) and the lyase is then eluted with a 0.3M phosphate buffer (pH 7.7).

Claim 14

A process according to claim 7, wherein the dialysis is carried out at a temperature of about 4° C..

Claim 15

A process according to claim 7, wherein the dialysis concentrate is dissolved in 0.1M aqueous sodium chloride solution and applied at about 4° C. to the cross-linked dextran gel which does not contain active carboxymethyl groups and which has been equilibrated with 0.1M aqueous sodium chloride solution.

Claim 16

A process according to claim 7, wherein the cross-linked dextran gel which does not contain active carboxymethyl groups is eluted with 0.1M aqueous sodium chloride solution.

Claim 17

A process according to claim 7, wherein the fractions from the treatment step with the cross-linked dextran gel which does not contain active carboxymethyl groups are dissolved in 0.1M acetate buffer (pH 5.0), applied at about 4° C. to a cross-linked dextran gel containing active carboxymethyl groups which has been equilibrated with 0.1M acetate buffer (pH 5.0) and the gel then eluted with 1.0M acetate buffer (pH 5.0).