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Bioconversion of cholesterol to 4‐cholesten‐3‐one by recombinant Bacillus subtilis expressing choM gene encoding cholesterol oxidase from Mycobacterium neoaurum JC ‐12
Author(s) -
Shao Minglong,
Rao Zhiming,
Zhang Xian,
Xu Meijuan,
Yang Taowei,
Li Hui,
Xu Zhenghong,
Yang Shangtian
Publication year - 2015
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.4491
Subject(s) - bioconversion , bacillus subtilis , cholesterol oxidase , recombinant dna , biochemistry , enzyme , chemistry , biology , microbiology and biotechnology , bacteria , gene , fermentation , genetics
BACKGROUND Bacillus subtilis 168 was used to express the cholesterol oxidase ( EC 1.1.3.6) genes cloned from Mycobacterium neoaurum JC ‐12 and the bioconversion of cholesterol to 4‐cholesten‐3‐one was realized. RESULTS Expression of cholesterol oxidase ChoM1 and ChoM2 from M. neoaurum in B. subtilis was confirmed by SDS‐PAGE and enzyme activity analysis. The activities of the recombinant ChoM1 and ChoM2 were 5.27 and 7.44 U mg −1 , which were 5.2‐fold and 7.3‐fold, respectively, of the cholesterol oxidases activities in M. neoaurum JC ‐12. Enzyme properties showed that the optimum pH and temperature for both ChoM1 and ChoM2 was 7.5 and 40 °C. Both enzymes were stimulated by Mg 2+ and Mn 2+ and showed higher activity towards the 3β‐hydroxy steroids with long alkyl chains at C17 . Whole‐cells of the recombinant strains were used as catalysts, the recombinant strains B. subtilis 168/ pMA5 ‐ choM1 and B. subtilis 168/ pMA5 ‐ choM2 catalyzed the bioconversion of cholesterol to 4‐cholesten‐3‐one with a percentage conversion of 67% and 83% at 21 h. CONCLUSION The recombinant strains demonstrated efficient and stable enzyme activities and showed obvious pre‐eminence on the bioconversion of cholesterol into 4‐cholesten‐3‐one. As a safe microorganism, B. subtilis 168 would be a promising cell factory to produce 4‐cholesten‐3‐one in the pharmaceutical industry. © 2014 Society of Chemical Industry