Isolation and Identification of A Native Rhodococcus Strain Producing Cholesterol Oxidase From Soil

Cholesterol oxidase (Cho) has various clinical and industrial applications. Recently, microbial Cholesterol oxidases have received a great attention for their wide usage in medical practice for determination of cell-free and membrane-bound type of Cholesterol. In this study different biochemical and microbiological tests were performed on isolated bacteria to identify the microorganism properties. For phylogenic analysis, a partial sequence of l6s rRNA was amplified by PCR using universally conserved primers. For determination of Cho activity, the modified method of Sasaki et al. was applied. In the next step, extra-cellular Cho activity was assayed under different temperature, pH and Cholesterol concentration conditions. At the end Cholesterol oxidase gene was amplified by PCR method and cloned into STV28 cloning vector. According to obtained results by morphological, cultural, biochemical and phylogenic tests, the isolated bacterium was identified as Rhodococcus sp. Strain 501 and was deposited in Gene bank. The results obtained from the effects of different physical parameters on Cho activity showed that the optimum temperature and pH for Cho production were found to be 35°C and 7.0, respectively. Nucleotide sequences of Cho gene was aligned and showed 99% homology with the other bacterial Cho genes. Rhodococcus sp. Strain 501 was able to produce significant levels of extra-cellular Cho in a short time period in the optimized medium which can be used as useful source for production of Cho in an industrial scale.