Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta.

Abstract

A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (COX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of COX by manipulation of various physicochemical parameters. The extracellular COX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified COX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105kDa), multimeric (pentamer of ∼21kDa protein) nature, organic solvent compatibility, and a half-life of ∼2h at 37°C. The bacterial COX exhibited ∼22% higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05% (v/v). The K m and V max value of COX of B. subtilis COX were found to be 3.25mM and 2.17μmolminml(-1), respectively. The purified COX showed very little cytotoxicity associated with it.