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

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

Biochemical composition of crustacean zooplankton and their grazing on phytoplankton and ciliated protozoans in a recently founded reservoir (Sahela, Morocco

In order to assess the impact of crustacean zooplankton on phytoplankton and protozoan ciliates in the Sahela reservoir under semi-arid climate, we conducted experiments during the period from July to December 1999 at the deepest point in the lake (15 m). Samplings and measurements were carried out in diffusion chambers submerged in situ over a period of 7 h without (control chambers) and with (experimental chambers) crustacean zooplankton. During these experiments, counts were conducted on phytoplankton and ciliates to determine the abundance and the mortality of these organisms due to zooplankton in each diffusion chambers at t = 0 and t = 7 h of incubation. The study showed that the growth rates of phytoplankton and ciliates populations varied between 0.02 and 0.05 h-1 and from 0.01 to 0.07 h-1, respectively. The mortality caused by zooplankton grazing fluctuated from 0.07 to 0.2 h-1 of phytoplankton and from 0.01 to 0.2 h-1 of ciliates. These mortalities were significantly and positively correlated with the growth rates (r = 0.8; p < 0.02; n = 9). Moreover, the heavy predation by the crustacean zooplankton was exerted on small-sized phytoplankton and ciliates and we demonstrated the relationships between protozoans and zooplankton for the transfer of matter and energy in aquatic food webs. Furthermore, the crustacean zooplankton metabolism was different, whether zooplankton was present in diffusion chambers or in the lake.