牛瘤胃内切葡聚糖酶基因在大肠杆菌中的克隆与表达  

Cloning and expression of the endoglucanase gene from cattle rumen in Escherichia coli

作　　者：马春娟 杨宇泽 邹爱爱 孙康永杰 万雪瑞 王川[1] 魏亚琴[3] MA Chunjuan;YANG Yuze;ZOU Aiai;SUN Kangyongjie;WAN Xuerui;WANG Chuan;WEI Yaqin(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,Gansu,China;Beijing Animal Husbandry Station,Beijing 100107,China;Key Laboratory of Microbial Resources Exploitation and Application of Gansu Province,Institute of Biology,Gansu Academy of Sciences/Center of Anaerobic Microbes,Lanzhou 730000,Gansu,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]北京市畜牧总站,北京100107 [3]甘肃省科学院生物研究所甘肃省微生物资源开发利用重点实验室/厌氧微生物中心,甘肃兰州730000

出　　处：《草业科学》2023年第4期1039-1047,共9页Pratacultural Science

基　　金：甘肃省科技计划项目重点研发计划(18YF1NA077、20YF3NA020);国家自然科学基金(31760614)。

摘　　要：为提高纤维素的降解效率、构建高效表达的纤维素酶基因工程菌,本研究以牛瘤胃液微生物全基因组为模板,首先通过PCR扩增内切葡聚糖酶eg基因,然后与pET-28a连接获得表达载体pET-28a::eg并转化至大肠杆菌(Escherichia coli)菌株BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达EG蛋白,最后用3,5-二硝基水杨酸(DNS)法测定重组内切酶的酶活力,分析其酶学性质。结果表明:成功构建表达载体pET-28a::eg,重组菌株E.coli BL21/pET-28a::eg在28℃用IPTG诱导14 h后纯化得到重组的EG蛋白,EG蛋白大小约为50 kDa,刚果红染色有明显水解圈。用DNS法测得EG的酶活为12.60 U·mL^(−1),滤纸总酶活为3.53 U·mL^(−1)。重组酶在不同底物的反应中,羧甲基纤维素钠为底物的酶活力最高,脱脂棉最低。重组酶的最适温度为40℃,最适pH为7.0。在此条件下,Ca^(2+)、Mg^(2+)、Fe^(2+)、K^(+)、Mn^(2+)等离子均可对重组蛋白EG的酶活力具有促进作用,Zn^(2+)可促进但差异不显著,而Hg^(2+)、Cu^(2+)对EG的酶活力具有抑制作用。本研究构建的重组内切酶菌株可以高效水解纤维素,为内切酶的工业应用奠定了基础。A high cellulose degradation efficiency is vital in industrial settings.In this study,we constructed an Escherichia coli strain overexpressing recombinant endoglucanase that can efficiently hydrolyze cellulose.Using the genome of bovine rumen juice microorganisms as a template,the endoglucanase eg gene was amplified by PCR.The PCR product was ligated to pET-28a to yield the expression vector pET-28a::eg.pET-28a::eg was transformed into E.coli strain BL21(DE3),and the expression of the EG protein in E.coli BL21/pET-28a::eg was induced by isopropyl-β-D-thiogalactopyranoside(IPTG).Finally,the enzymatic activity of the recombinant endoglucanase was measured using the 3,5-Dinitrosalicylic acid(DNS)method,and its enzymatic properties were determined.The results showed that the expression vector pET-28a::eg was successfully constructed.EG expression was induced with IPTG in E.coli BL21/pET-28a::eg at 28℃for 14 h.The molecular weight of the recombinant EG protein was approximately 50 kDa after purification,with a hydrolysis circle visible with Congo red staining.The enzyme activity of EG is 12.60 U·mL^(−1) and the total enzyme activity of filter paper is 3.53 U·mL^(−1).Among the reactions of recombinase with different substrates,the enzyme activity towards sodium CMC-Na was the highest,and the activity towards absorbent cotton was the lowest.The optimal temperature for the recombinant enzyme was 40℃,and the optimal pH was 7.0.Under these conditions,Ca^(2+),Mg^(2+),Fe^(2+),K^(+),and Mn^(2+)plasma all contributed to the enzymatic activity of EG.While Zn2+also contributed to EG activity,the contribution was not significant,whereas Hg^(2+)and Cu^(2+)inhibited enzyme activity.The construction of a bacterial strain harboring recombinant endoglucanase capable of efficiently degrading cellulose lays the foundation for the industrial application of endoglucanases.

关 键 词：内切葡聚糖酶 eg基因 大肠杆菌 克隆 蛋白表达 酶活 酶学性质 

分 类 号：Q78[生物学—分子生物学] S816[农业科学—饲料科学]