Cholesterol oxidase (COX, E.C.22.214.171.124), 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.