Purification and characterization of Chromobacterium sp. DS-1 cholesterol oxidase with thermal, organic solvent, and detergent tolerance.
A new screening method for 6beta-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0-7.5, showing optimal activity at pH 7.0 and 65 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.
Doukyu
Noriyuki
N
Bio-Nano Electronic Research Center, Toyo University, 2100 Kujirai, Kawagoe, Saitama, Japan. dokyu@toyonet.toyo.ac.jp
Shibata
Kanpei
K
Ogino
Hiroyasu
H
Sagermann
Martin
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000077463:Carbamide Peroxide; D002784:Cholesterol / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002861:Chromobacterium / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004338:Drug Combinations; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010545:Peroxides / Q000378:metabolism; D010802:Phylogeny; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D014508:Urea / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; D004338:Drug Combinations; D010545:Peroxides; D012997:Solvents; D000077463:Carbamide Peroxide; D014508:Urea; D002784:Cholesterol; D002789:Cholesterol Oxidase