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

2002; Oxford University Press; Volume: 215; Issue: 2 Linguagem: Inglês

10.1111/j.1574-6968.2002.tb11397.x

1574-6968

Kinya Fujishiro, Hiroyuki Uchida, Kotaro Shimokawa, Maki Nakano, Fumihiko Sano, Toshio Ohta, Norihiko Kayahara, Kazuo Aisaka, Takayuki Uwajima,

Hormonal Regulation and Hypertension

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).