Purification, characterization, and biological cytotoxic activity of the extracellular cholesterol oxidase produced by Castellaniella sp. COX.

A new bacterial strain producing extracellular cholesterol oxidase (ChOx) was isolated and identified as Castellaniella sp. COX. The ChOx was purified by salting-out and ion-exchange chromatography up to 10.4-fold, with a specific activity of 15 U/mg with a molecular mass of 59 kDa. The purified ChOx exhibited pH 8.0 and temperature 40°C for its optimum activity. The enzyme showed stability over a wide pH range and was most stable at pH value 7.0, and at pH 8.0, it retained almost 86% of its initial activity after 3 h of incubation at 37°C. The enzyme possessed a half-life of 8 h at 37°C, 7 h at 40°C, and 3 h at 50°C. A Lineweaver-Burk plot was calibrated to determine its Km (0.16 mM) and Vmax (18.7 μmol·mg-1 ·min-1 ). The ChOx activity was enhanced with Ca2+ , Mg2+ , and Mn2+ while it was inhibited by Hg2+ , Ba2+ , Fe2+ , Cu2+ , and Zn2+ ions. Organic solvents like acetone, n-butanol, toluene, dimethyl sulfoxide, chloroform, benzene, and methanol were well tolerated by the enzyme while iso-propanol and ethanol were found to enhance the activity of purified ChOx. ChOx induced cytotoxicity with an IC50 value of 1.78 and 1.88 U/ml against human RD and U87MG established cell lines, respectively, while broadly sparing the normal human cells.