Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

摘要: Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation homogeneous as judged sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. molecular weight of the enzyme estimated to be 53,000 SDS-gel electrophoresis and 46,000 sedimentation equilibrium. contained 483 amino acid residues calculated on basis 53,000. consumed 60 mumol O2/min per mg protein with 1.3 mM cholesterol at 37 degrees C. showed highest activity cholesterol; 3 beta-hydroxysteroids, such dehydroepiandrosterone, pregnenolone, lanosterol, were also oxidized slower rates. Ergosterol not enzyme. Km for 0.33 optimal pH 5.0. is a flavoprotein which shows visible absorption spectrum having peaks 353 nm 455 in 0.1 M acetate buffer, 4.0. characterized hypsochromic shift second peak bound flavin. flavin reduced anaerobic addition model substrate, dehydroepiandrosterone. Neither heat treatment released coenzyme protein. could easily acid-soluble form peptides when digested trypsin plus chymotrypsin. mobilities aminoacyl after hydrolysis thin layer high voltage differed those free FAD, FMN, riboflavin. A pKa value 5.1 obtained pH-dependent fluorescence quenching process AMP detected nucleotide pyrophosphatase. results indicate strongly that contains FAD prothetic group, covalently linked properties comparable N (1)-histidyl FAD.