Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli.
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 degrees C in Luria-Bertani medium containing 50 microM 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-choB(b).
Wang
Longgang
L
Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Southern Yangtze University, Wuxi, 214036, PR China. wanglongang2004@yahoo.com.cn
Wang
Wu
W
D000595:Amino Acid Sequence; D001951:Brevibacterium / Q000201:enzymology*; D002789:Cholesterol Oxidase / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects*; D008969:Molecular Sequence Data; D012256:Riboflavin / Q000494:pharmacology*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D002789:Cholesterol Oxidase; D012256:Riboflavin