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Cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp.

Authors: Hamed Esmaeil Lashgarian, Kiana Shahzamani and Sareh Jahanbakhsh

Int J Med Res Health Sci.91-96 | pdf PDF Full Text

Cholesterol oxidase (CHO) is one of the valuable enzymes that play an important role in: measurement of serum
cholesterol, food industry as a biocatalyst and agriculture as a biological larvicide. This enzyme was produced by
several bacterial strains. Wild type enzyme produced by Rhodococcus sp. secret two forms of CHO enzyme: extra
cellular and membrane bound type which its amount is low and unstable. The goal of the study was cloning,
expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp.
CHO gene was isolated from native bacteria and cloned into pET23a. In the next step, the construct was expressed
in E.coli BL21 and induced by different concentration of IPTG ranges from 0.1 – 0.9 mM. This gene contains 1642
bp and encodes a protein consists of 533 amino acids. It has about 96 % homology with CHO gene isolated from
Rhodococcus equi. The high expression was obtained in 0.5 mM concentration of IPTG after 4 hour induction. This
recombinant enzyme had a molecular weight of 55 kDa, that secretion of intra cellular type is much more than extra
cellular form. The optimum pH and temperature conditions for the recombinant enzyme were 7.5 and 45°C,
respectively. CHO enzyme obtained from Rhodococcus sp. is a cheap enzyme with medical and industrial
applications that can be produced easily and purified in large scale with simple methods.

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