Cholesterol oxidase

A method for quantitative determination of cholesterol contained in a sample which comprises:

• exposing the sample to a cholesterol oxidase to oxidize the cholesterol, and
• measuring the amount of oxidized cholesterol or hydrogen peroxide thereby determining the amount of cholesterol,
• wherein the cholesterol oxidase has the amino acid sequence as defined in the Sequence Listing by SEQ ID NO:
• 4, and the following physicochemical properties:
• (a) activity which catalyzes the reaction of oxidizing cholesterol in the presence of oxygen to form hydrogen peroxide and 4-cholesten-3-one;
• (b) an isoelectric point at a pH of 4.7;
• (c) substrate specificity so that the enzyme acts on cholesterol, .beta.-sitosterol, stigmasterol, pregnenolone, dehydroisoandrosterone and estradiol but does not act on vitamin D.sub.3, cholic acid, androsterone, cholesterol linoleate or lanosterol;
• (d) a working pH in a range of 5.0 to 7.5, and the enzyme is stable in a pH range of 5.3 to 7.5 when heated at 50.degree. C. for 60 minutes;
• (e) optimum temperature of about 50.degree. C.;
• (f) the enzyme is inactivated at a pH of 10.0 or more or at pH of 4.0 or less when heated at 50.degree. C. for an hour and the enzyme is also inactivated by about 83% when heated at a pH of 7.0 and a temperature of 60.degree. C. for an hour;
• (g) the enzyme is inhibited by p-chloromercury benzenesulfonate, silver nitrate and o-hydroxyquinoline and in the presence of bovine serum albumin, resistance to heat and stability during storage are improved;
• (h) Michaelis' constant to cholesterol (Km value) of 3.0.times.10.sup.-5 M;
• and
• (i) molecular Weight of about 43,000 (gel filtration).