Some enzymatic properties of 3beta-hydroxysteroid oxidase produced by Streptomyces violascens

Abstract

The 3beta-hydroxysteroid oxidase produced by Streptomyces violascens was purified from the culture broth by procedures including batch-wise treatment on DEAE-cellulose, ammonium sulfate fraction, gel filtration on Sephadex G-75, and column chromatography on DEAE-cellulose. The highly purified enzyme preparation exhibited no significant absorption maxima in the visible region other than a maximum at 280 nm. Optimum pH and temperature for the enzyme activity were approximately pH 7.5 and 50 degree, respectively. The Michaelis constant (Km) for cholesterol determined under two different experimental conditions were 4.5 and 6.7 X 10(-4) M. The enzymatic activity was remarkably inhibited by various metal salts such as FeC1(3), FeSO4, AgNO3, etc. On the other hand, neither EDTA nor Fe-chelating agents had any inhibitory effect on the enzymatic activity, while other metal-binding agents, KCN and NaN3, caused significant inhibition. The enzyme activity was inhibited almost completely by N-bromosuccinimide and iodine but not p-chloromercuribenzoate. The highly purified enzyme did not require any external electron acceptors other than oxygen. In addition, the activity was not influenced by the addition of external electron donors. The enzyme showed a high substrate specificity for 3beta-hydroxysteroids and the relative oxidation rates were 100 for cholesterol, 91 for 5alpha-cholestan-3beta-ol, 83 for pregn-5-en3beta-ol-one, 80 for androst-5-en-3beta-ol-17-one, 64 for 5alpha-androstan-3beta-ol-17-one, ect. The oxidation of cholesterol by the enzyme was remarkably inhibited by the addition of 5alpha-cholestan-3beta-ol, 5alpha-cholestan-3-one, 5beta-cholestan-3beta-ol, 5alpha-cholestane3beta, 5alpha-doil or 5alpha-lanosta-8, 24-den 3beta-ol. These findings indicate the present enzyme belongs to the class of 3beta-hydroxysteroid oxidase but some of its physical and enzymatic properties obviously differ from those of 3beta-hyroxysteroid oxidase of Brevibacterium.