Membrane-bound cholesterol oxidase from Rhodococcus sp. cells. Production and extraction

Abstract

Evidence was demonstrated for the occurrence of a membrane-bound cholesterol oxidase (EC 1.1.3.6) in cells of Rhodococcus sp. GK1, a soil-isolated strain. This enzyme catalyzed the conversion of 3 β-OH- Δ 5-sterols to their 3-Keto- Δ 4 derivatives with concomitant reduction of O 2 to H 2O 2. Growth of the microbe in a mineral medium, containing either phytosterols or hexanoate as sole carbon substrate, was studied. Under these conditions, the membrane-bound cholesterol oxidase was induced in large amounts exceeding 100 U g −1 dry wt. Enzyme solubilization was achieved by cell treatment with either Triton X-100, Emulphogen BC-720, Lubrol PX, Lubrol WX, or Brij 76, all are nonionic detergents and have low critical micelle concentration (CMC). Enzyme solubilization was due to mixed micelle formation. An additional evidence for the mixed micelle formation was provided from both, enzyme filtration on Sephadex G-200 in the presence of Triton X-100, and enzyme activity stimulation by nonionic detergents. This type of cholesterol oxidase was found to be an integral membrane protein. It appeared that the enzyme active domain is located at the outer surface of the membrane. Substrate catalysis in vivo occurs externally to the cytoplasm. A topology model at the membrane level was suggested for cholesterol oxidases of Rhodococcus and related taxa. The detergent-extracted cholesterol oxidase was active on sterols with the 3 β-OH- Δ 5 configuration. Neither sterols with modified A-ring or B-ring nor the 3α-OH of cholic acid were catalyzed by it. With cholesterol as the substrate, the optimal pH was in a broad region, from 6.0 to 8.2 at 30°C, and the K m value was 1.9 × 10 −5M at pH 7.0 and 30°C. With β-sitosterol, a K m of 2.1 × 10 −5M was estimated for the enzyme under similar conditions. Preparations of the detergent-extracted enzyme were stable upon maintenance at either 2–4°C for 6 months or at −20°C for 1 year.