植物乳杆菌DPP8胆盐水解酶基因在大肠杆菌中的表达及其酶学性质

丁轲; 段锦; 余祖华; 李旺; 李元晓; 何万领; 张春杰; 丁盼盼; 王玉琴; 刘宁

doi:10.3969/j.issn.1006-267x.2020.07.043

动物营养学报 >

2020 , Vol. 32 >Issue 7: 3333 - 3342

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.07.043

分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

植物乳杆菌DPP8胆盐水解酶基因在大肠杆菌中的表达及其酶学性质

丁轲 ,
段锦 ,
余祖华 ,
李旺 ,
李元晓 ,
何万领 ,
张春杰 ,
丁盼盼 ,
王玉琴 ,
刘宁

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1. 河南科技大学宏翔生物饲料实验室, 洛阳 471000;
2. 河南省动物疫病与公共卫生重点实验室, 洛阳 471000;
3. 河南省肉羊繁育工程技术研究中心, 洛阳 471000

丁轲(1977-),男,河南永城人,副教授,博士,主要从事动物微生态与动物传染病学研究。E-mail:keding19@163.com

收稿日期: 2020-01-28

网络出版日期: 2020-07-15

基金资助

河南省自然科学基金项目（182300410052）；河南省科技攻关项目（182102110184）

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Expression of Bile Salt Hydrolase Gene from Lactobacillus plantarum DPP8 in Escherichia coli and Its Enzymatic Properties

DING Ke ,
DUAN Jin ,
YU Zuhua ,
LI Wang ,
LI Yuanxiao ,
HE Wanling ,
ZHANG Chunjie ,
DING Panpan ,
WANG Yuqin ,
LIU Ning

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1. Hongxiang Biological Feed Laboratory, Henan University of Science and Technology, Luoyang 471000, China;
2. Key Laboratory of Animal Disease and Public Health of Henan Province, Luoyang 471000, China;
3. Research Center of Breeding Engineering Technology for Meat Sheep of Henan Province, Luoyang 471000, China

Received date: 2020-01-28

Online published: 2020-07-15

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摘要

本试验旨在克隆植物乳杆菌DPP8胆盐水解酶（BSH）基因并在大肠杆菌表达系统对其进行表达，同时探讨其酶学性质。通过PCR技术克隆植物乳杆菌DPP8 BSH基因，然后将该基因重组到大肠杆菌表达载体pET32a（+）中，获得重组表达载体pET32a（+）-BSH，再将其转化至大肠杆菌BL21中，通过异丙基硫代半乳糖苷（IPTG）诱导表达BSH。采用牛磺酸标准液标定法对表达的BSH的活性进行测定，并对其酶学性质进行研究。结果显示：本试验获得了BSH基因全长975 bp的序列，同源性比较和系统进化树分析表明成功获得了BSH基因。对BSH表达产物的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）显示获得了1条约为56 ku的蛋白条带，与预期蛋白分子质量大小相符。重组菌株在37℃、0.1 mmol/L IPTG诱导6 h时BSH活性最高，可达22.81 U/mL。纯化的BSH的最适反应温度为37℃，最适反应pH为6.5，在40℃以下时具有较好的热稳定性，pH在3.0~9.0时可保持90%以上的活性。镁离子（Mg²⁺）、铝离子（Al³⁺）、三价铁离子（Fe³⁺）对BSH活性具有较强的抑制作用，而钠离子（Na⁺）、钾离子（K⁺）、二价铁离子（Fe²⁺）、铜离子（Cu²⁺）、锰离子（Mn²⁺）、锌离子（Zn²⁺）、钙离子（Ca²⁺）对BSH活性无明显抑制作用。综上，本试验成功表达了植物乳杆菌DPP8 BSH，且其活性可达22.81 U/mL，在40℃以下和pH 3.0~9.0时能够保持较强的活性，但Mg²⁺、Al³⁺和Fe³⁺对其活性具有较强的抑制作用。

关键词： 植物乳杆菌; 胆盐水解酶; 克隆; 表达; 酶学性质

本文引用格式

丁轲 , 段锦 , 余祖华 , 李旺 , 李元晓 , 何万领 , 张春杰 , 丁盼盼 , 王玉琴 , 刘宁 . 植物乳杆菌DPP8胆盐水解酶基因在大肠杆菌中的表达及其酶学性质[J]. 动物营养学报, 2020 , 32(7) : 3333 -3342 . DOI: 10.3969/j.issn.1006-267x.2020.07.043

Abstract

The aim of this experiment was to clone the bile salt hydrolase (BSH) gene from Lactobacillus plantarum DPP8 and express it in Escherichia coli (E. coli) expression system, and to study its enzymatic properties. The BSH gene was cloned by PCR technique, and was inserted into vector pET32a(+) to construct the recombinant expression vector pET32a(+)-BSH, which was transformed into E.coli BL21. The recombinant strain was induced to express BSH by isopropylthiogalactosidehe (IPTG). The expressed BSH activity was detected by calibration method of taurine standard solution, and the enzymatic properties of the expressed BSH was studied. The results showed that a whole 975 bp sequence was obtained, and was also proved successfully that it was the BSH gene by homology comparison and phylogenetic tree analysis. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay indicated that the colecular weight of the recombinant protein was approximately 56 ku, which was consistent with the expected size. The highest BSH activity was 22.81 U/mL in recombinant strain with the conditions at 37℃, 0.1 mmol/L IPTG induction 6 h. The reaction optimum temperature of the purified BSH was 37℃ and the enzyme can still maintain the good thermal stability under 40℃ for 1 h. The reaction optimum pH of the purified BSH was 6.5 and more than 90% enzyme activity was maintained at pH 3.0 to 9.0, 37℃ for 1 h. Mg²⁺, Al³⁺ and Fe³⁺ had strong inhibition effects on the BSH activity, but Na⁺, K⁺, Fe²⁺, Cu²⁺, Mn²⁺, Zn²⁺ and Ca²⁺ had no obvious effects on it. In conclusion, the BSH from Lactobacillus plantarum DPP8 is expressed in E. coli, successfully, the highest BSH activity is 22.81 U/mL, and it can keep high enzyme activity under 40℃ and within pH 3.0 to 9.0, but can be strong inhibited by Mg²⁺, Al³⁺ and Fe³⁺.

Key words： Lactobacillus plantarum; bile salt hydrolase; clone; expression; enzymatic properties

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