Purification and Characterization of Cholesterol Oxidase from a Novel Source - Enterobacter cloacae

The renewed interest in exploring various natural habitat/environments for discovering novel microbial sources as stable cholesterol oxidase producers is on the incline due to its broad-range of clinical and industrial applications. A search was conducted to isolate cholesterol oxidase producing bacteria from samples collected from different environments. A total of 98 bacterial isolates capable of degrading cholesterol were obtained from various soil samples collected from in and around Chennai. The bacterial strain producing the highest level of cholesterol oxidase was selected, identified as Enterobacter cloacae by molecular studies and used for all further studies. Purification of cholesterol oxidase was carried out by ammonium sulphate precipitation (80% w/v) followed by Q sepharose and DEAEcellulose chromatographie techniques. The enzyme was purified to 4.63 fold with a recovery of 93.8 mg of purified protein from 2,889 mg of total protein. The purification yield of the enzyme was 14.86% and its specific activity was 21.8 Umg. SDS PAGE and MALDI TOF analyses proved that the enzyme was a monomer with a molecular mass of 56.50461. The enzyme exhibited optimum activity at pH 7.0 and was stable up to 40C for an hour. Metal ions such as, Mn, K and Zn inhibited the enzyme activity by 82, 37.66 and 32.8% respectively while most of the other metal ions showed less than 25% inhibition. Triton X-100 and Tween-80 failed to inhibit its activity, however SDS decreased in its activity by 35.2%. Therefore E.cloacae can be exploited for industrial production of stable cholesterol oxidase.