变形杆菌脂肪酶在大肠杆菌细胞表面的展示表达及重组酶的特性  

Display expression of a lipase from Proteus sp. on the Escherichia coli cell surface and characterization of the recombinant enzyme

作　　者：田淑芳 王雨 李春华 TIAN Shufang;WANG Yu;LI Chunhua(Hubei Key Laboratory of Industrial Biotechnology, Hubei Collaborative Innovation Center for Green Transformation of Bio resources,College of Life Sciences,Hubei University,Wuhan 430062,China))

机构地区：[1]湖北大学生命科学学院,生物资源绿色转化湖北省协同创新中心,工业生物技术湖北省重点实验室,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2018年第1期1-6,共6页Journal of Hubei University：Natural Science

基　　金：国家自然科学基金(31100124);湖北省教育厅重点项目(D20131004)资助

摘　　要：在生物转化处理工业中利用细胞表面展示技术表达脂肪酶作为一种全细胞催化形式具有很大优势,因为脂肪酶展示在细胞表面作为一种固定化酶.以超折叠绿色荧光蛋白的羧基端结构域作为锚定区,构建变形杆菌来源的脂肪酶与羧基端结构域的融合基因.将融合基因插入到p ETsf GFP表达载体中,得到表面展示载体p ETsf GFP-Lip A.将重组质粒转入大肠杆菌Rosetta-Blue宿主菌中经自诱导后,利用免疫荧光显微技术和流式细胞仪分析确定脂肪酶成功展示于大肠杆菌细胞表面.重组酶的最高酶活达到7 009 U/g干细胞,最适反应温度和p H分别为为37℃和8.0,表面活性剂Tirton X-100、Co^(2+)和Ni^(2+)能显著提高脂肪酶的酶活,60%丙酮处理该酶18 h后脂肪酶残余酶活性为104%,60%乙醇处理该酶18 h后脂肪酶残余酶活性为117%,表明脂肪酶对有机试剂具有很好的耐受性.以上实验结果表明:细胞表面展示技术表达脂肪酶的最大优点为工业化应用提供了坚实的基础.Background for industrial bioconversion processes,the utilization of surface displayed lipase in the form of whole cell biocatalysts is more advantageous,because the enzymes are displayed on the cell surface spontaneously,regarded as immobilized enzymes.The carboxyl terminal domain of sfGFP(super fold green fluorescent protein)was used as carrier protein and the LipA gene from Proteus sp.was fused to the sfGFP gene.The fusion gene sfGFP LipA was inserted the plasmid pETsfGFP,which was named as pETsfGFP LipA.The recombinant plasmid was transformed into E.coli Rosetta Blue host after self induced expression.Localization of the displayed LipA on the cell surface was confirmed by the confocal laser scanning microscopy and flow cytometry.The activity of the recombinant LipA has reached to7009U/g dry cells,and the optimal temperature and pH of lipase was37℃and8.0,respectively.The activity was significantly increased in the presence of the metal ions such as Co2+and Ni2+and Tirton X100.It was indicated that the recombinant LipA exhibited resistance to organic solvents after the treatment with60%acetone and60%ethanol for18h,the activity was increased by104%and117%,respectively.So the above results indicate that the advantage of surface displayed lipase could lay a solid foundation for industrial application.

关 键 词：表面展示 融合表达 脂肪酶 全细胞催化剂 

分 类 号：Q78[生物学—分子生物学]