De Forenede Bryggerier A/s

Carlsberg Biotechnology Ltd. A/s

De Forende Bryggerier A/s

De Forenede Bryggeier A/s

Jack T Johansen

Fred Widmer

Klaus Breddam

Process for enzymatic production of peptides

Process for enzymatic replacement of the B-30 amino acid in insulins

An insulin such as porcine insulin is reacted enzymatically with an L-amino acid, amide, or ester in the presence of L-specific serine carboxypeptidase modified by reaction with divalent metal ions in aqueous solution or dispersion containing F.sup.-, Cl.sup.-, Br.sup.-, I.sup.-, CN.sup.-, or SCN.sup.-.

Cu,Zn-superoxide dismutase (SOD) is isolated from aqueous solutions containing said enzyme together with accompanying proteins by chromatography of the solution at a pH of 4.7 to 5.0 on a cation exchange resin of the same polarity as SOD in the pH range used. As cation exchange resin may particularly be used carboxymethyl celluloses, cross-linked dextrans substituted with carboxymethyl groups or sulfopropyl groups or cross-linked agaroses substituted with carboxymethyl groups. The process lends itself to use on an industrial scale and provides a high yield of pure SOD.

Process for isolating Cu, Zn-superoxide dismutase from aqueous solutions

Cu,Zn-superoxide dismutase (SOD) is isolated from aqueous solutions containing said enzyme together with accompanying proteins by chromatography of the solution at a pH of 4.7 to 5.5 on a cation exchange resin of the same polarity as SOD in the pH range used. As cation exchange resin may particularly be used carboxymethyl celluloses, cross-liked dextrans substituted with carboxymethyl groups or sulfopropyl groups or cross-linked agaroses substituted with carboxymethyl groups. The process lends itself to use on an industrial scale and provides a high yield of pure SOD.

Process for isolating Cu,Zn-superoxide dismutase from aqueous solutions

Various enzymes, in particular Cu,Zn-superoxide dismutase (SOD), catalase and carbonic acid anhydrase, are recovered from blood by admixing wholly or partly isolated blood cells with ethanol or a homologous alcohol until a concentration of 10 to 70% by volume and allowing them to stand for hemolysis of the blood cells and denaturation of the hemoglobin, and then adding water up to the double volume or more, and removing the precipitate of cell residuals, hemoglobin and other denatured proteins from the suspension. Then the desired enzymes are isolated from the solution obtained. In particular, SOD and catalase can both be isolated by chromatography of the solution at a pH of 4.7 to 5.5 on a cation exchange resin of the same polarity as SOD in the pH range used and elution of the resin with a buffer solution which has a pH in the range 4.7 to 7.5 and an ionic strength in the range 0.01 to 1.0 M, SOD being eluted at the lowest pH and/or the lowest ionic strength. The process allows both the hemolysis of the blood cells and the precipitation of the hemoglobin to be carried out in one step while retaining the activity of the desired enzymes, so that they can be obtained in a high yield in a manner which lends itself to use on an industrial scale. Further, SOD and catalase may be isolated and purified in a single chromatography step.

Process for recovering enzymes from blood

Cu,Zn-superoxide dismutase is recovered from yeast by plasmolysis with a small amount of ether or any other water immiscible, organic solvent and subsequent autolysis in water at a temperature of 25.degree. to 50.degree. C. and pH 5 to 9, following which the precipitate is removed and the superoxide dismutase is purified and isolated from the residual liquid, in particular by chromatography on carboxymethyl cellulose at pH 4.7 to 5.5. Cu,Zn-superoxide dismutase from Saccharomyces cerevisiae has the amino acid sequence: ##STR1## where Cys-57 and Cys-146 form a disulfide bond.

Growing community of inventors

Rungsted Kyst, Denmark

Jack T Johansen