Secretory expression and characterization of a bile salt hydrolase from Lactobacillus plantarum in Escherichia coli
Secretory expression and characterization of a bile salt hydrolase from Lactobacillus plantarum in Escherichia coli
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植物乳杆菌胆汁盐水解酶在大肠杆菌中的分泌表达和表征
DOI：
10.1016/j.molcatb.2013.04.006
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发表时间：
2013-09-01
影响因子：
--
通讯作者：
Chen, Jian
中科院分区：
文献类型：
--
作者：
Dong, Zixing;Zhang, Juan;Chen, Jian
由于pET载体的PelB信号肽（分泌系统）不能有效分泌胆盐水解酶（BSH），我们使用双精氨酸易位（达特）途径作为这种分泌产生的替代途径。在这里，我们报告的BSH分泌的双精氨酸信号肽的二甲基亚砜（DMSO）还原酶亚基DmsA从大肠杆菌。虽然表达的蛋白质大部分是胞内包涵体，但也有一些蛋白质被成功地分泌到培养基中。当使用Ni-NTA亲和层析从上清液中纯化BSH至均一时，通过SDS-PAGE估计分子量为37.0 kDa。当pH和温度最适为pH 6.0和37 ℃时，pH和热稳定性分别为pH 7.0-8.0和40 ℃。底物特异性研究表明，这种酶是更有效地水解糖结合胆汁盐比牛磺结合胆汁盐。以甘氨脱氧胆酸盐（GDCA）为底物，米氏常数（Km）为2.07 mM，最大反应速率（Vmax）为142.8 μ mol/（min mg），转换数（Kcat）为88.127 s（-1）。二硫苏糖醇（DTT）和乙二胺四乙酸（EDTA）可增强BSH活性，而高碘酸钠和CuCl 2则完全抑制BSH活性。据我们所知，这是第一个报告的分泌表达BSH的双精氨酸信号肽，生化特性的研究将奠定理论基础，BSH的结构分析。(C)2013爱思唯尔有限公司版权所有。
As PelB signal peptide (secretory system) of pET vectors was not effective in secreting bile salt hydrolase (BSH), we used the twin-arginine translocation (Tat) pathway as an alternative for this secretory production. Here we report the BSH secretion by the twin-arginine signal peptide of dimethyl sulfoxide (DMSO) reductase subunit DmsA from Escherichia coli. Although most of the proteins expressed were intracellular inclusion bodies, some proteins were successfully secreted to the medium. When BSH was purified to homogeneity from the supernatant using Ni-NTA affinity chromatography, the molecular mass was estimated to be 37.0 kDa by SDS-PAGE. While the pH and temperature optima were at pH 6.0 and 37 degrees C, the pH and thermal stability were pH 7.0-8.0 and 40 degrees C, respectively. Substrate specificity studies showed that this enzyme is more efficient in hydrolyzing glycoconjugated bile salts than tauro-conjugated bile salts. With glycodeoxycholate (GDCA) as the substrate, the Michaelis constant (K-m) and maximum reaction rate (V-max) were determined to be 2.07 mM and 142.8 mu mol/(min mg), respectively, and the turnover number (K-cat) was 88.127 s(-1). BSH activity was enhanced by dithiothreitol (DTT) and ethylene diamine tetraacetic acid (EDTA), and completely inhibited by sodium periodate and CuCl2. To the best of our knowledge, this is the first report on the secretory expression of BSH using twin-arginine signal peptides, and the biochemical characteristics investigated would lay a theoretical foundation for the structure analysis of BSH. (C) 2013 Elsevier B.V. All rights reserved.