title: Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.
abstract: Cholesterol oxidase production (COD) by a new isolate characterized as Streptomyces sp. was studied in different production media and fermentation conditions. Individual supplementation of 1 % maltose, lactose, sucrose, peptone, soybean meal and yeast extract enhanced COD production by 80-110 % in comparison to the basal production medium (2.4 U/ml). Supplementation of 0.05 % cholesterol (inducer) enhanced COD production by 150 %. COD was purified 14.3-fold and its molecular weight was found to be 62 kDa. Vmax (21.93 μM/min mg) and substrate affinity Km (101.3 μM) suggested high affinity of the COD for cholesterol. In presence of Ba(2+) and Hg(2+) the enzyme activity was inhibited while Cu(2+) enhanced the activity nearly threefold. Relative activity of the enzyme was found maximum in triton X-100 whereas sodium dodecyl sulfate inactivated the enzyme. The enzyme activity was also inhibited by the thiol-reducing reagents like Dithiothreitol and β-mercaptoethanol. The COD showed moderate stability towards all organic solvents except acetone, benzene and chloroform. The activity increased in presence of isopropanol and ethanol. The enzyme was most active at pH 7 and 37 °C temperature. This organism is not reported to produce COD.
authors.affiliation: Division of Fermentation Technology, CSIR-Central Drug Research Institute, Chattar Manzil, PO Box 173, Lucknow, 226001, India.
authors.forename: Ram
authors.initials: R
authors.lastname: Niwas
authors.forename: Vineeta
authors.initials: V
authors.lastname: Singh
authors.forename: Rajbir
authors.initials: R
authors.lastname: Singh
authors.forename: Divya
authors.initials: D
authors.lastname: Tripathi
authors.forename: C K M
authors.initials: CK
authors.lastname: Tripathi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D002789:Cholesterol Oxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005285:Fermentation; D010084:Oxidation-Reduction; D010802:Phylogeny; D012988:Soil Microbiology; D013302:Streptomyces / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D002784:Cholesterol; D002789:Cholesterol Oxidase