Characterization of a secreted cholesterol oxidase from Rhodococcus sp.GKI (CIP 105335)

摘要: Extracellular cholesterol oxidase (COX) (EC 1.1.3.6) was produced by Rhodococcus sp. GK1 cells grown in a defined mineral salt medium containing mixture of phytosterols (sitosterol, campesterol, stigmasterol) as the sole source carbon and energy. In same time, sterols acted enzyme inducers. The enriched with yeast extract order to stimulate secretion. COX purified from culture supernatants affinity-like chromatography on column packed kieselguhr cholesterol. Enzyme bound onto eluted 0.05 M phosphate buffer pH 7.0 Triton X-100 at 0.1% (w/v). Some properties were determined. Its specific activity 30 °C, around 5.5 units mg−1. molecular mass enzyme, estimated SDS-PAGE, 59 kDa. isoelectrofocusing point 8.9. C-5 double bond alkyl chain moiety sterol molecules necessary for an adequate oxidation 3β-ol. inhibition ions (0.1 mM): AsO2−, Ba2+, Co2+, Cd2+, Cu2+, N3−, Ni2+, Pb2+ negligible (around 10%). However, 0.1 mM either Zn2+, 2-[(ethylmercurio)-thio]benzoic acid, or Hg2+ 18%, 22% 93% respectively. Inhibition significant, even 1 μM. (0.1–0.15 mg ml−1 7.0) relatively heat-stable temperatures up 50 °C. At this temperature, half-life its 70 min. 90% initial lost 20 min incubation 60 aminoacid sequence N-terminal segment was: H2N–Ala–Pro–Pro–Val–Ala–Ser–X–Arg–Tyr–X–(Phe)– (X might be 2 Cys residues).