Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli |
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Authors: | Longgang Wang Wu Wang |
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Affiliation: | (1) Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Southern Yangtze University, Wuxi, 214036, P.R. China |
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Abstract: | The gene (choB b ), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs and encodes a protein of 551 amino acids. ChoB b exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia coli BL21–CodonPlus (DE3)-RP grown at 23°C in Luria-Bertani medium containing 50 μM riboflavin, the precursor of the FAD coenzyme of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choBb. |
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Keywords: | Brevibacterium sp. Cholesterol oxidase Escherichia coli Over-expression Riboflavin |
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