SEPARATION AND PURIFICATION OF 3-OXO-5BETA-STEROID-DELTA4-DEHYDROGENASE

PURPOSE:To obtain high purity 3-oxo-5beta-steroid-DELTA -dehydrogenase (3-OSD) in separated and purified state on an industrial scale, by adding a specific gel to a solution containing 3-OSD, thereby allowing the gel to adsorb 3-OSD. CONSTITUTION:Polyvinyl gel or agarose gel having bonded butyl group is suspended in a salt buffer solution of about 7pH at a concentration of 10-30wt% and the suspension is filled in a column. A 3-OSD solution produced by dissolving 3-OSD in a buffer solution having an electrical conductivity of 50-100mmho/cm and a definite pH is filled in the column to effect the adsorption of 3-OSD to the gel. A buffer solution same as the above buffer solution is passed through the column at an amount corresponding to 2-3 times the volume of the column to elute impurities. After the above treatment, a buffer solution of 7pH having low salt concentration is passed through the column to elute the enzyme. High purity 3-OSD produced by this process has a purification ratio of as high as 21-35 based on the crude raw material.