Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10

• Published in: FEMS microbiology letters Vol. 215; no. 2; pp. 243 - 248
• Main Authors: Fujishiro, K, Uchida, H, Shimokawa, K, Nakano, M, Sano, F, Ohta, T, Kayahara, N, Aisaka, K, Uwajima, T
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.10.2002; Blackwell; Oxford University Press
• Subjects: absorption; Bacteriology; beta-sitosterol; Biological and medical sciences; Brevibacterium; Brevibacterium - enzymology; Brevibacterium - genetics; Brevibacterium sterolicum; Cholesterol; Cholesterol oxidase; Cholesterol Oxidase - chemistry; Cholesterol Oxidase - genetics; Cholesterol Oxidase - isolation & purification; Cholesterol Oxidase - metabolism; Chromatography, High Pressure Liquid; Cloning; Cloning, Molecular; Colony hybridization; E coli; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli MM294; Expression cloning; flavoproteins; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; genes; Hydrogen-Ion Concentration; Hydroxysteroids; Metabolism. Enzymes; Microbiology; molecular weight; New species; pH effects; Pregnenolone; Purification; Substrate Specificity; temperature; Cholesterol oxidase; Expression cloning; Colony hybridization; MM294; Cholesterol; Purification; Enzyme; Escherichia coli; Oxidase; Brevibacteriaceae; Enzymatic activity; Bacteria; Escherichia coli MM294; Actinomycetes; Oxidoreductases; Recombinant protein; Brevibacterium sterolicum; Enterobacteriaceae
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2002.tb11397.x

A gene encoding a cholesterol oxidase from Brevibacterium sterolicum novo sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55 degrees C, respectively. The enzyme acted on 3 beta-hydroxysteroids such as cholesterol, pregnenolone, and beta-sitosterol at high rates, but on dehydro-epi-androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum novo sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (K(m) = 30 micromolar).