Abstract
Cholesterol oxidases, which catalyze the degradation of cholesterol to cholest-4-en-3-one, are widely used in the pharmaceutical and food processing industries. The cholesterol oxidase from Pimelobacter simplex (PsChO3) was transformed into E. coli BL21(DE3), but it was expressed mainly as inclusion bodies, and any soluble PsChO3 failed to bind to Ni-NTA resin. To overcome this obstacle, we devised a simple yet efficient purification and refolding process using 8 M urea for the solubilization of PsChO3 and achieved a high yield of the enzyme in its active form. Column-bound PsChO3 was refolded in situ through a gradient of successively decreased urea concentrations and purified using Ni-affinity chromatography, ionic exchange and gel filtration. This treatment converted the denatured PsChO3 into a soluble protein exhibiting an unexpected dehydrogenation activity amounting to 9.27 U/mg - an activity not reported for enzymes with noncovalently-linked FAD to date. The product, cholest-5-en-3-one, was confirmed using TLC, GC-MS and NMR. Structural analysis revealed a distinct binding mode in both FAD and substrate domain, which may explain the enzyme's unusual catalytic behavior.
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