Purification and characterization of extracellular cholesterol oxidase from Enterobacter sp.

The objective of this work is to obtain an abundant source of cholesterol oxidases for industrial and medicinal needs. Thirteen bacterial strains that express high level of inducible extracellular cholesterol oxidase (COX) were isolated from carnivore feces. One of these strains, named COX8-9, belonging to the genus Enterobacter, was found to produce the highest level of cholesterol oxidase. COX from strain COX8-9 was purified from the culture supernatant by ultrafiltration followed with two consecutive Q-Sepharose chromatographies at different pH values, and then by Superdex-75 gel filtration. The purified enzyme was a monomer with a molecular weight of 58 kDa, and exhibited maximum absorption at 280 nm. The Km value for oxidation of cholesterol by this enzyme was 1.2 × 10−4 M, with optimum activity at pH 7.0. Enzymatic activity of COX was enhanced 3-fold in the presence of metal ion Cu2+, and the enzyme was stable during long-term aqueous storage under various temperatures, indicating its potential as a clinical diagnostic reagent. Preparation and characterization of cholesterol oxidases from the other selected strains are under way.