A Research Paper: Screening, Characterization and Structural Modeling of Extracellular Cholesterol Oxidase Isolated from Malaysian Rhodococcus Isolates
Purpose: This project was initiated to screen the ability of Malaysian Rhodococcus isolates in producing Cholesterol Oxidase (CHO) and to characterize the enzyme activity.
Background: CHO (EC 22.214.171.124) is an industrially importance enzyme which catalyzes the cholesterol degradation to 4-cholestene-3-one by oxygen reduction. This enzyme is widely used in the determination of cholesterol in blood serum as well as in food and is gaining a high demand from the industry. Many microorganisms were reported to be able to produce this enzyme with various level of production.
Design/Methodology/Approach: Twenty three Rhodococcus spp. supplied by Unisel Culture Collection (UCC) were screened for their ability to produce CHO using the selective agar with cholesterol as the only carbon source. The ability to degrade cholesterol was measured by the capability of the isolates to grow on the selective. The strain with the highest growth on the selective agar was then selected for enzyme activity characterization. 16S rRNA sequencing was done for species identification and to establish the phylogenetics map of the 23 isolates. Study of the effect of fermentation parameters on the enzyme activity was done by on-factor at a time approach to determine the optimal incubation conditions for CHO production.
Results/Findings: All 23 isolates were observed to be able to grow on the selective agar. UCC0021 was seen as the best cholesterol degrader with the highest density of cell growth on the plate and was selected for further study. The identity of the isolates were determined using the 16S rRNA sequences obtained and phylogenetics tree showing the evolutionary relationship of the 23 isolates was established. The highest enzyme activity produced was 1.364 U/mL observed at 108 h of incubation in initial fermentation conditions. The optimization study showed the optimal culture conditions at temperature 35 °C, pH 9.5, Tween 80 2.5 mL/L, Triton-X 100 2.5 mL/L, NaCl 0.5 mg/L, cholesterol 3 mg/L, 150 rpm and 25% (v/v) of inculum size producing the highest enzyme activity of 1.506 U/mL at 96 h incubation.
Conclusion and Implications: The study has successfully revealed that all Malaysian Rhodococcus isolates are able to degrade cholesterol and Rhodococcus strain UCC0021 has a great potential to be utilized for CHO enzyme production. CHO produced may has great implication in food quality especially in poultry industry where this enzyme could be used as an agent to reduce cholesterol level in egg yolk.