Purification and some properties of cholesterol oxidase stable in detergents from γ-proteobacterium Y-134

Cholesterol oxidase (CHO) with high stability in detergents was found from an isolated strain, Y-134, belonging to the γ-subclass of Proteobacteria. CHO production reached its maximum by incubation at 30°C for 12 d. It was purified from cell-free extract prepared by mixing the cells with 0.4% Triton X-100. The absorption spectrum of the purified enzyme exhibited maxima at 274 and 410 nm, and a shoulder at 330 nm. The molecular mass was 115 kDa with two identical subunits of 58 kDa. The enzyme oxidized cholest-5-en-3β-ol (cholesterol) and 5α-cholestan-3β-ol (dihydrocholesterol) at a high reaction rate, and the K m value for cholesterol was 65 μM. The stability of the enzyme was higher than other CHOs in nonionic detergents with high values of hydrophilelipophile balance (HLB) such as Triton X-450 and sodium cholate. NH 2-terminal sequence analysis showed a high similarity to CHO from Burkholderia cepacia, but not to CHOs from Streptomyces or Brevibacterium.