Ahmad, Seraj2015-10-192023-10-192015-10-192023-10-192015ROLL NO. 08610616https://gyan.iitg.ac.in/handle/123456789/617Supervisor: Pranab GoswamiCholesterol oxidase (EC 1.1.3.6) is a flavoenzyme that in most cases catalyses the oxidation of cholesterol to 4-cholesten-3-one (CEO, cholestenone). The product cholestenone has been used as a precursor for the synthesis of hormones and many intermediate steroidal compounds which are eventually used to produce anabolic drugs and contraceptive. Classical (one-variable-at-a-time) and statistical methods (Plackett-Burman and Central composite design) were used to optimize growth medium for the production of cholesterol oxidase from Rhodococcus sp. NCIM 2891. Cholesterol oxidase activities from the classically and statistically optimized media were 0.75 and 3.25 U/ml, respectively. The statistically optimized medium had 4.33- and 9.7-fold higher enzymatic activity than the classically optimized and unoptimized basal medium, respectively. The ratio of enzyme production to cell growth rate was 29-fold higher in our statistically optimized medium than in the basal medium, indicating that the enzyme production could be classified as mixed type of growth. Cell-bound cholesterol oxidase accounted for 90.68±2 % of the total enzymatic activity of the growth medium. Interactions between the cholesterol oxidase-inducing substrate cholesterol and medium growth substrates yeast extract and (NH4)2HPO4 significantly enhanced the production of cell bound cholesterol oxidase. Our results validate the statistical approach as a potential technique for achieving the large-scale production of cell-bound cholesterol oxidase from Rhodococcus sp. NCIM 2891. The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified 3-fold by Diethylaminoethyl-Sepharose chromatography. The estimated molecular mass (SDS-PAGE) and Km of the enzyme were ~ 55.0 kDa and 151 μM, respectively. 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 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, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. The both free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ~ 88 % milli molar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ~ 67 % activity even after twelve successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.enBIOSCIENCES AND BIOENGINEERINGThesis