Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from Pseudomonas sp. Strain ST-200

Extracellular cholesterol oxidase of Pseudomonas sp. strain ST-200 was purified from the culture supernatant. This oxidase contained bound flavin and was categorized as a 3β-hydroxysteroid oxidase, converting 3β-hydroxyl groups to keto groups. The molecular mass was 60 kDa. The enzyme was stable at pH 4 to 11 and active at pH 5.0 to 8.5, showing optimal activity at pH 7 at 60°C. The Michaelis constant of the ST-200 cholesterol oxidase was lower than those of commercially available oxidases. The cholesterol oxidation rate was enhanced 3- to 3.5-fold in the presence of organic solvents, with log P(ow) values (partition coefficients of the organic solvent between n-octanol and water), in the range of 2.1 to 4.2, compared with that in the absence of organic solvents.