Purification and Characterization of Extracellular 3β-Hydroxysteroid Oxidase produced by Streptoverticillium cholesterolicum

Abstract

3β-Hydroxysteroid oxidase activity was detected in the broth filtrate of strain H 1109 MY 12. Based on taxonomic studies, this strain was shown to be a new species of genus Streptoverticillium and the name Stv. cholesterolicum is proposed for this strain. The enzyme was purified by ammonium sulfate precipitation and affinity column chromatography on crystalline cholesterol, and the product showed a single band on SDS-polyacrylamide gel electrophoresis. The enzyme showed optimum activity at pH 7.0-7.5 and was stable over a rather wide pH range of 4.0 to 12.5. Cholesterol and dihydrocholesterol were oxidized rapidly. The K_m value for the oxidation of cholesterol by this enzyme was about 0.4mM. The enzyme activity was greately inhibited by HgCl₂, and AgNO₃. FeCl₃ and CuSO₄ were also inhibitory though to lesser extents. Iodine and N-bromosuccinimide completely inhibited the enzyme activity at concentrations of less than 0.01 mM. Neither metal-binding agents nor p-chloromercuribenzoic acid had any inhibitory effect on the oxidation of cholesterol by this enzyme. The molecular weight of the enzyme was estimated to be 56000 by SDS-polyacrylamide gel electrophoresis. The enzyme was proved to be a flavoprotein containing flavin adenine dinucleotide as a prosthetic group.