Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2

Rhodococcus sp. R14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH2-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a rather wide pH range of 4.0¿10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly oxidizes 3ß-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3¿-hydroxysteroids. Thus, the presence of a 3ß-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated at 55 ¿M; the COX activity was markedly inhibited by metal ions such as Hg2+ and Fe3+ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione. These suggests that -SH groups may be involved in the catalytic activity of the present COX