Bioconversion of cholesterol to 4‐cholesten‐3‐one by recombinant Bacillus subtilis expressing choM gene encoding cholesterol oxidase from Mycobacterium neoaurumJC‐12

Abstract

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 Mg2+ and Mn2+ 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

Enhanced intracellular soluble production of 3‐ketosteroid‐Δ1‐dehydrogenase from Mycobacterium neoaurum in Escherichia coli and its application in the androst‐1,4‐diene‐3,17‐dione production

“…The activity is expressed as U mg −1 of protein (the crude protein of the special fraction of cell extract), and 1 U is defined as the reduction of 1 µmol min −1 DCPIP (ξ 600 = 11.3 × 10 3 cm −1 M −1 ). The SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis of the different protein fractions was according to our previous work …”

“…The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the different protein fractions was according to our previous work. 19…”

Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ1-dehydrogenase and catalase inBacillus subtilis

“…; Shao et al . ). Therefore, during the sterol microbial transformation process, there was H 2 O 2 accumulated which might have toxic effects on sterol bioconversion.…”

Heterologous expression and characterization of a 3-ketosteroid-∆1-dehydrogenase from Gordonia neofelifaecis and its utilization in the bioconversion of androst-4,9(11)-dien-3,17-dione

“…The degradation of cholesterol or its derivatives begins with the transformation of cholesterol to cholest-4-en-3-one by a cholesterol oxidase (Shao et al 2015). The subsequent catabolism involves elimination of the alkyl side chain followed by the opening of the rings A/B and rings C/D.…”