Enhanced Production of Cholesterol Oxidase from Bacillus sp. COX-T<sub>3</sub> in a Biphasic System

Abstract

Cholesterol oxidase (COX, E.C.1.1.3.6), catalyses the oxidation of cholesterol to 4-cholesten-3-one with the reduction of oxygen to hydrogen peroxide. Qualitative analysis of bacterial strain (Bacillus sp. BT COX-T3) gave Gram positive rod-shaped colonies with high cholesterol degradation. Organic solvent tolerant microorganisms are novel group of extremophilic microorganisms that have developed resistance to withstand solvent toxicity. These organisms play an important role in biotransansformation and bioconversion of organic compounds. An organic solvent-tolerant Bacillus sp. BT COX-T3 obtained after primary screening for production of extracellular COX transformed cholesterol to 4-cholesten- 3-one in toluene, and resulted in a higher COX production in a biphasic medium due to an increase in solubility of cholesterol in the medium making it easily available to be metabolized by the organism. A simple screening method for 6β-hydroperoxycholest-4-en-3-one formed by Bacillus sp. COX-T3 was devised in this study and bioconversion was monitored spectrophotometerically. Presence of cholesterol in 20% (v/v) toluene was observed to be most effective biotransformation system for bacterial COX production. A maximum COX activity of 0.892 U/ml was obtained at 45°C by using 8% (v/v) inoculum, 0.1% (w/v) cholesterol as the main carbon source and 0.5% (w/v) yeast extract. Amongst various salt ions only Co2+ ions improved the COX production by Bacillus sp. COX-T3. Moreover, the cholesterol provided in the bioconversion system was completely transformed by Bacillus sp. COX-T3. Bacillus sp COX-T3 effectively degraded cholesterol in the presence of toluene and the extracellular COX produced by it was highly stable at the end of 144 h. Keywords: Cholesterol, cholesterol oxidase, bioconversion, solvent tolerant.