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

Cholesterol oxidase (CHO) with high stability in detergents was found from an isolated strain, Y-134, belonging to the gamma-subclass of Proteobacteria. CHO production reached its maximum by incubation at 30 degrees 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-3beta-ol (cholesterol) and 5alpha-cholestan-3beta-ol (dihydrocholesterol) at a high reaction rate, and the K(m) value for cholesterol was 65 microM. 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. NH2-terminal sequence analysis showed a high similarity to CHO from Burkholderia cepacia, but not to CHOs from Streptomyces or Brevibacterium.