分子营养 Molecular Nutrition

2个枯草芽孢杆菌源纤维素酶基因的克隆、融合表达及其酶学性质分析

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  • 1. 河南科技大学宏翔生物饲料实验室, 洛阳 471023;
    2. 河南省动物疫病与公共卫生重点实验室, 洛阳 471023;
    3. 河南省肉羊繁育工程技术研究中心, 洛阳 471023
丁轲(1977—),男,河南永城人,副教授,博士,主要从事动物微生态学研究。E-mail:keding19@163.com

收稿日期: 2017-02-08

  网络出版日期: 2017-08-02

基金资助

国家自然科学基金项目(31101744);河南省科技厅重大科技攻关项目(131100110300)

Cloning, Fusion Expression of Two Cellulase Genes from Bacillus subtilis and Its Enzymatic Properties

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  • 1. Hongxiang Biological Feed Laboratory, Henan University of Science and Technology, Luoyang 471023, China;
    2. Key Laboratory of Animal Disease and Public Health of Henan Province, Luoyang 471023, China;
    3. Research Center of Breeding Engineering Technology for Meat Sheep of Henan Province, Luoyang 471023, China

Received date: 2017-02-08

  Online published: 2017-08-02

摘要

本试验旨在构建不同纤维素酶的融合表达系统及探讨融合纤维素酶的酶学性质。利用PCR技术从实验室前期分离的枯草芽孢杆菌中分别扩增2个纤维素酶基因Cel42和Cel22,设计一段柔性接头(GSGGGS),通过酶切连接将2个纤维素酶基因构建在一个开放阅读框(ORF)内,插入到pET32a(+)中构建重组表达载体pET32a(+)-Cel42-Cel22,转化大肠杆菌BL21(DE3)进行诱导表达,并对其酶学性质进行研究。结果表明:本试验成功克隆了2个纤维素酶基因Cel42和Cel22,并构建了重组表达系统BL21(DE3)/pET32a(+)-Cel42-Cel22,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)估计其分子质量约为101 ku,粗酶液中葡聚糖内切酶活性为57.62 U/mL,葡聚糖外切酶活性为32.57 U/mL。试验所得融合纤维素酶Cel42-Cel22的最适反应温度为50 ℃,最适反应pH为6.0,温度在30~70 ℃范围内时可维持70%以上的纤维素酶活性,pH在4.0~9.0范围内时可保持75%以上的纤维素酶活性,除Mn2+外的其他金属离子对纤维素酶的活性均具有一定的抑制作用,其中Hg2+和Cu2+对的抑制作用较明显。由此可见,本试验在大肠杆菌BL21(DE3)中成功表达出了融合纤维素酶Cel42-Cel22,且该酶具有一定的活性,可适应较宽广的温度和pH范围,对金属离子敏感。

本文引用格式

丁轲, 邱静静, 罗伟光, 李旺, 李元晓, 曹平华, 何万领, 赵龙妹, 王玉琴, 张春杰 . 2个枯草芽孢杆菌源纤维素酶基因的克隆、融合表达及其酶学性质分析[J]. 动物营养学报, 2017 , 29(8) : 2725 -2733 . DOI: 10.3969/j.issn.1006-267x.2017.08.015

Abstract

The aim of this experiment was to construct fusion expression system based on different cellulases and studied the enzymatic properties of fusion cellulase. The two different cellulase genes Cel42 and Cel22 were amplified from Bacillus subtilis isolated in previous researches in our laboratory using PCR method, respectively. The two genes were linked with a flexible polypeptide (GSGGGS), which could form a complete ORF, and the fusion gene was inserted into vector pET32a(+) to construct the recombinant expression vector pET32a(+)-Cel42-Cel22, which was transformed into Escherichia coli BL21(DE3). The recombinant strain was induced to express the fusion cellulose, and the the enzymatic properties of fusion cellulose were studied. The results showed that the two cellulase genes Cel42 and Cel22 were cloned successfully in this experiment, and obtained the recombinant expression system BL21/pET32a(+)-Cel42-Cel22. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the molecular weight of the fusion protein was about 101 ku. Enzymatic activity of the crude enzyme liquid indicated that the endo-1,4-β-D-glucanases activity was 57.62 U/mL, and the exo-1,4-β-D-glucanase activity was 32.57 U/mL. The reaction optimal temperature of the fusion cellulase Cel42-Cel22 was 50 ℃, and could still maintain above 70% cellulase activity when temperature was from 30 to 70 ℃. The optimal pH of the fusion cellulase Cel42-Cel22 was 6.0, and could still maintain over 75% cellulase activity in a pH range of 4.0 to 9.0. In addition to Mn2+, other metal ions had inhibitory effects on the activity of fusion cellulase Cel42-Cel22, especially Hg2+ and Cu2+. In conclusion, the fusion fusion cellulase Cel42-Cel22 is realized to efficiently express in Escherichia coli BL21(DE3), has a higher activity within a wide range of temperature and pH, and is sensitive to metal ions.

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