GB1067253A - Process for the preparation of enzymes - Google Patents

Abstract

In a process for the production of very pure chondroitinase, cells of Proteus vulgaris (N.C.T.C. 4636) are macerated with a solvent, filtered off and dried, the dried material homogenized in a buffer solution with a pH of about 7.0, incubated at this pH for a short time, the liquid phase separated and dialysed against water, the dialysed, cell-free extract mixed with an aqueous solution of a salt of yeast nucleic acid previously adjusted to a pH of about 7.4, the material which thereby precipitates re-suspended in water and dissolved by the addition of an aqueous solution of an alkali metal hydroxide until the pH is about 7.4, an aqueous solution of protamine sulphate added until the addition of protamine sulphate to a sample clarified by centrifuging no longer gives a precipitate, a slight excess of protamine sulphate then added and the solution as a whole dialysed against water, precipitated material removed, the supernatant liquid adjusted to a pH of about 8.0, cooled, clarified and again dialysed, the resultant clear solution finally being lyophilized. The powder obtained may be adsorbed on calcium phosphate gel and fractionally eluted with sodium acetate, or if it is desired to recover also the chondrosulphatase with which the chondroitinase is contaminated, the two enzymes may be separated chromatographically on DEAE-"Sephadex" (Registered Trade Mark).