Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

Abstract

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 Km of the enzyme were ∼55.0kDa and 151μM, 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°C, 5min) for activity of the enzyme was increased by 5°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 0.1mM of each acted as inhibitors, while Ag+, Ca2+, Ni2+ and Zn2+ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75mM) with the cholesterol oxidase immobilized beads (3.50U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9h at 25°C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.