Efficient production of active recombinant cholesterol oxidase from Rhodococcus erythropolis in Escherichia coli via his-tag assisted refolding strategy for cholesterol oxidation

This study reports the cloning, expression, and characterization of recombinant cholesterol oxidase (ChOx) from Rhodococcus erythropolis in Escherichia coli BL21(DE3). The ChOx gene was inserted into the pET23b(+) vector using primers designed with site-specific modifications that enabled His-tag activation, expression, and purification by nickel-affinity chromatography to yield a protein of ∼55 kDa. A urea-gradient dialysis refolding protocol was employed to recover active enzyme from inclusion bodies. The purified enzyme exhibited a specific activity of 13.68 U/mg, representing a 2.5-fold improvement over previously reported recombinant ChOx yields with a recovery yield of 76.16 %. Cholesterol oxidation assays optimized enzyme concentration, substrate concentration, and temperature. Optimum activity occurred at 0.4 mg/mL enzyme and 20 mM cholesterol, achieving > 80 % substrate conversion, while the enzyme retained stability up to 50 °C. This work demonstrates a His-tag-assisted refolding strategy that efficiently produces active recombinant Rhodococcus erythropolis ChOx (RhoChOx). The optimization of catalytic parameters highlights the enzyme's robustness under diverse conditions. These findings establish RhoChOx as a promising biocatalyst for industrial cholesterol oxidation and provide a methodological framework applicable to other recombinant oxidases.