辅酶前体辅助短小芽孢杆菌来源的胆固醇氧化酶在大肠杆菌中的表达

Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli.

作者信息

Wang Longgang, Wang Wu

机构信息

Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Southern Yangtze University, Wuxi, 214036, PR China.

出版信息

Biotechnol Lett. 2007 May;29(5):761-6. doi: 10.1007/s10529-006-9295-0. Epub 2007 Jan 20.

DOI:10.1007/s10529-006-9295-0

PMID:17237971

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 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).

摘要

克隆并测序了来自短杆菌属CCTCC M201008的编码胆固醇氧化酶的基因（choB(b)）（通过PCR技术，GenBank登录号：DQ345780）。该基因由1653个碱基对组成，编码一个含551个氨基酸的蛋白质。ChoB(b)与来自嗜固醇短杆菌ATCC 21387的胆固醇氧化酶基因具有98%的同源性。克隆到载体pET-28a中的胆固醇氧化酶基因，在含有50微摩尔核黄素（该酶FAD辅酶的前体）的Luria-Bertani培养基中，于23℃培养的大肠杆菌BL21-CodonPlus(DE3)-RP中实现了过量表达。从携带pET-28a-choB(b)的大肠杆菌BL21-CodonPlus(DE3)-RP的无细胞提取物中获得了最高活性为3.7 U/mg的酶。