牛瘤胃纤维素酶eg基因在乳酸菌中的克隆表达及酶学性质分析  

Expression of the eg gene of cellulase from cattle rumen in Lactobacillus and analysis of enzymatic properties

作　　者：邹爱爱 魏亚琴[2] 杨宇泽 曹磊 孙康永杰 万学瑞[1] 王川[1] ZOU Aiai;WEI Yaqin;YANG Yuze;CAO Lei;SUN Kangyongjie;WAN Xuerui;WANG Chuan(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,Gansu,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;Beijing Animal Husbandry Station,Beijing 100107,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]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 [3]北京市畜牧总站,北京100107

出　　处：《草业科学》2021年第12期2471-2480,共10页Pratacultural Science

基　　金：甘肃省科技计划项目重点研发计划(18YF1NA077);甘肃农业大学动物医学院学科建设基金(GSAU-XKJS-2018-074);甘肃省科学院科技产业化项目“牦牛瘤胃厌氧真菌基因重组工程菌发酵生产木质纤维素降解酶的研究”;甘肃农业大学2020年学生科研训练计划(SRTP202003018)。

摘　　要：为构建高效表达的内切葡聚糖酶基因工程菌,本研究以牛瘤胃液中微生物全基因组为模板,通过PCR扩增方法得到eg片段,将其克隆至表达载体pMG36e中获得分泌型表达载体pMG36e::eg;将测序正确后的重组质粒电转导到乳酸菌(Lactococcus lactis NZ9000)中,得到L.lactis NZ9000/pMG36e::eg重组菌株,将发酵上清液通过10%三氯乙酸(Trichloroacetic acid,TCA)/丙酮沉淀法浓缩蛋白后,用刚果红染色法和3,5-二硝基水杨酸(3,5-Dinitrosalicylic acid,DNS)法检测该蛋白酶活性,使用滤纸酶活力(filter paper enzyme activity,FPA)法检测该蛋白酶的总酶活,并对重组内切葡聚糖酶进行酶学性质研究。结果表明:从牛瘤胃微生物中克隆得到一个基因大小为1500 bp左右的条带,该酶分子量为50 kDa左右,经刚果红染色有明显水解圈,水解圈直径为2.32 cm;采用DNS法测定该重组蛋白的酶活为12.4019 U·mL^(−1),用FPA法测定总酶活为12.2469 U·mL^(−1);重组蛋白酶对羧甲基纤维素钠、滤纸、微晶纤维素、脱脂棉均有酶活性;最适反应温度为90℃;最适反应pH为6;其中Cu^(2+)、Mn^(2+)、Ba^(2+)、Zn^(2+)、Co^(2+)等均可以提高重组酶的酶活力,而Fe^(2+)可抑制重组内切葡聚糖酶活力。本试验纤维素酶eg基因在L.lactis NZ9000中的稳定高效表达为提高青贮饲料的营养价值和消化率提供了技术支持。In order to construct highly expressing endoglucanase genetically engineered bacteria,the whole genome of the microorganism in bovine rumen juice was used as a template in this study,and the eg fragment was obtained by PCR amplification In order to construct genetically engineered bacteria with high endoglucanase expression,we used the whole genomes of microorganisms in bovine rumen juice as a template.We obtained a fragment containing the eg gene of cellulase by PCR amplification,which was cloned into the expression vector pMG36e to yield the expression vector pMG36e::eg.Recombinant plasmids containing the pMG36e::eg construct were electrotransduced into lactic acid bacteria(Lactococcus lactis NZ9000)to obtain an L.lactis NZ9000/pMG36e::eg recombinant strain,and the fermentation supernatant of the recombinant strain was concentrated using the 10%trichloroacetic acid/acetone precipitation method.Enzyme activity of the recombinant endoglucanase was determined using the 3,5-dinitrosalicylic acid(DNS)and Congo red staining methods,total enzyme activity was determined using the filter paper enzyme activity(FPA)method,and enzymatic properties were examined.A gene of approximately 1500 bp was cloned from a bovine rumen microorganism and the molecular weight of the encoded enzyme was approximately 50 kDa.Congo red staining analysis revealed that the recombinant enzyme caused a clear 2.32 cm zone of hydrolysis.The enzyme activity of the recombinant protein was 12.4019 U·mL^(−1 )based on the DNS method and 12.2469 U·mL^(−1 )using the FPA method.Furthermore,the recombinant protease has enzymatic activity on CMC-Na,filter paper,microcrystalline cellulose,and absorbent cotton.Enzyme activity was found to be optimal at a temperature of 90°C and pH 6.Metal ions,including Cu^(2+),Mn^(2+),Ba^(2+),Zn^(2+),and Co^(2+),were found to promote activity of the recombinant enzyme,whereas Fe^(2+)inhibited recombinant endoglucanase activity.In this study,we thus demonstrated the stable high-efficiency expression of the cellulase eg

关 键 词：内切葡聚糖酶 eg基因 乳酸菌 克隆 蛋白表达 酶活 酶学性质 

分 类 号：S816.3[农业科学—饲料科学]