The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl-sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K-m of the enzyme were similar to 55.0 kDa and 151 mu M, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 degrees C, 5 min) for activity of the enzyme was increased by 5 degrees C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe2+ and Pb2+ at 0.1 mM of each acted as inhibitors, while Ag+, Ca2+, Ni2+ and Zn2+ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to similar to 88% millimolar yield of cholestenone in a reaction time of 9 h at 25 degrees C. The immobilized enzyme retains similar to 67% activity even after 12 successive batches of operation, The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone. (C) 2014 Elsevier Ltd. All rights reserved.