甲醇芽孢杆菌凝乳酶的重组表达及其结构特性  

Recombinant Expression and Structural Properties of Milk-Clotting Enzyme from Bacillus methanolicus

作　　者：李柳[1] 郑喆[1] 吴凤玉[1] 郝一江[1] 赵笑[1] 曹永强 余志坚 陈超 杨贞耐[1,2] LI Liu;ZHENG Zhe;WU Fengyu;HAO Yijiang;ZHAO Xiao;CAO Yongqiang;YU Zhijian;CHEN Chao;YANG Zhennai(Beijing Advanced Innovation Center for Food Nutrition and Human Health,Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients,Beijing Technology&Business University(BTBU),Beijing 100048,China;Dongjun Dairy(Yucheng)Co.Ltd.,Yucheng 251200,China)

机构地区：[1]北京食品营养与人类健康高精尖创新中心,食品添加剂与配料北京高校工程研究中心,北京工商大学,北京100048 [2]东君乳业(禹城)有限公司,山东禹城251200

出　　处：《食品科学》2019年第22期39-46,共8页Food Science

基　　金：国家自然科学基金青年科学基金项目(31601488)

摘　　要：为进一步了解微生物凝乳酶的结构特性,根据GenBank数据库中甲醇芽孢杆菌凝乳酶(I3EB99)的氨基酸序列和大肠杆菌密码子的偏爱性,设计合成此凝乳酶的全基因序列,构建原核表达载体,通过BL21(DE3)表达其融合蛋白,将获得的融合蛋白进行His标签特异性亲和纯化,并利用生物信息学方法研究凝乳酶的三维空间立体结构。结果表明,重组表达的甲醇芽孢杆菌凝乳酶质量浓度为0.7 mg/mL,凝乳活力为(15870±1.17)SU/g,蛋白水解活力为(263.81±0.94)U/g,凝乳活力与蛋白水解活力比值为60.16,符合干酪生产加工的要求。结构特性研究表明,经重组表达后的甲醇芽孢杆菌凝乳酶呈现疏水特性,具有跨膜结构和信号肽,二级结构中α-螺旋少于β-折叠,在分离纯化过程中结构不稳定易降解,该凝乳酶与来自甲醇芽孢杆菌的一种未知蛋白酶是同源蛋白,高级结构与PDB蛋白数据库中的模板蛋白2ra1.1.A相似度最高。通过对甲醇芽孢杆菌凝乳酶结构特性的研究,为深入分析该凝乳酶作用机理及其功能性奠定了理论基础。In this study,in order to further understand the structural properties of microbial milk clotting enzyme(MCE),we firstly designed the whole gene sequence of MCE(I3EB99)from Bacillus methanolicus according to the amino acid sequences obtained from the GenBank and the codon preference of Escherichia coli,and then constructed a prokaryotic expression vector.The fusion protein was expressed in E.coli BL21(DE3),and it was subjected to His-tag specific affinity purification.The three-dimensional spatial structure of the purified MCE was studied by bioinformatics methods.Results showed that the recombinant MCE was expressed at a level 0.7 mg/mL with milk clotting activity(MCA)of(15870±1.17)SU/g,proteolytic activity(PA)of(263.81±0.94)U/g and MCA/PA ratio of 60.16,which met the requirements for cheese processing.Studies on its structural properties showed that the MCE exhibited hydrophobicity and it contained a transmembrane structure and a signal peptide;theα-helix secondary structure was less abundant thanβ-sheet in the MCE,which was unstable and easily degraded during the separation and purification process.The MCE was homologous to an unknown protease from B.methanolicus,and its three-dimensional structure had the highest similarity to the template protein 2ra1.1.A in the PDB protein database.By studying the structural characteristics of MCE from B.methanolicus,we have established the theoretical basis for an in-depth understanding of the mechanism and function of the MCE.

关 键 词：凝乳酶I3EB99 全基因合成 原核表达 亲和纯化 结构预测 

分 类 号：TS252.5[轻工技术与工程—农产品加工及贮藏工程]