Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum

Abstract

The characterization, extraction, and purification of a cholesterol:oxygen oxidoreductase (EC 1.1.3.6) from Nocardia sp. is described. This enzyme catalyzes oxidation of cholesterol to Δ4 cholestenone, with production of hydrogen peroxide. It is very stable, active over a wide pH range, and has a K(m) of 1.4 x 10-5 mol/l. It is highly specific for Δ4 or Δ5 3β hydroxycholestanes, and may be applied to the assay of serum total cholesterol. In the procedure presented here, hydrogen peroxide is measured by reaction with quadrivalent titanium and xylenol orange. This constitutes a one enzyme assay with stable reagents, which does not require protein precipitation and is not subject to interference from hemoglobin or bilirubin.