title: Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10.
abstract: A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. 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 3beta-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 nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (K(m)=30 microM).
authors.affiliation: Research and Development Section, Kyowa Medex Co., Ltd., Sumitomo Irifune Building, 2-1-1 Irifune, Chuo-ku, Tokyo 104-0042, Japan.
authors.forename: Kinya
authors.initials: K
authors.lastname: Fujishiro
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Uchida
authors.forename: Kotaro
authors.initials: K
authors.lastname: Shimokawa
authors.forename: Maki
authors.initials: M
authors.lastname: Nakano
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Sano
authors.forename: Toshio
authors.initials: T
authors.lastname: Ohta
authors.forename: Norihiko
authors.initials: N
authors.lastname: Kayahara
authors.forename: Kazuo
authors.initials: K
authors.lastname: Aisaka
authors.forename: Takayuki
authors.initials: T
authors.lastname: Uwajima
mesh_terms: D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics; D002789:Cholesterol Oxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity
chemical_list: D002789:Cholesterol Oxidase