Purification of 3.BETA.-hydroxysteroid oxidase of Streptomyces violascens origin by affinity chromatography on cholesterol.

Abstract

Cholesterol could be used to adsorb the 3.beta.-hydroxysteroid oxidase of S. violascens origin from a crude enzyme solution, and the adsorbed enzyme could be eluted with a suitable detergent such as Triton X-100. The enzyme was purified by (NH4)2SO4 precipitation and affinity chromatography on cholesterol with a recovery of 79% from the culture filtrate. The purified enzyme was detected as a single band on SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis. The MW of the enzyme was 61,000 by SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibited a characteristic spectrum of flavoenzyme. The flavin moiety of the enzyme was isolated and identified as FAD.