摘要
利用IPTG诱导含有川芎咖啡酸-3-O-甲基转移酶(LCCOMT)的大肠杆菌工程菌E.coli BL21(DE3)/pET28a-LCCOMT,经Ni^(2+)亲和层析、质谱鉴定获得纯化的LCCOMT。采用海藻酸钙凝胶包埋固定LCCOMT,单因素实验考察最佳固定化条件对固定化酶活力的影响,并确定了固定化酶的最适温度、pH值、Km、Vmax与反应批次等酶学性质。确定的条件分别为海藻酸钠质量分数1.5%,CaCl_2浓度2.5 g/L,固定化时间2 h,载体与酶质量比40 000∶1。通过正交实验确定最终固定化条件为CaCl_2浓度2.5 g/L,海藻酸钠质量分数2.0%,固定化时间1 h,载体与酶质量分数35 000∶1时,固定化效果最好,此时相对酶活力为75.43%。固定化酶的最适催化温度为37℃、最适pH值为7.5,较游离酶分别增加0℃和0.5;Km、Vmax分别增高0.40和0.74;实验确定固定化酶的半衰期,连续使用6次,酶活力仍保留50%。
The recombinant Chuanxiong caffeic acid-3-O-methyltransferase(LCCOMT)was induced by IPTG from E.coli BL21(DE3)containing pET28a-LCCOMT,and purified by Ni 2+affinity column chromatography and identified by mass spectrometry.The recombinant LCCOMT was immobilized by calcium alginate gel.The optimal immobilization conditions,including optimum temperature,pH value,Km and Vmax,and the effect of reaction batch on the activity of immobilized enzyme were investigated by single factor experiment.The determined conditions were as follows∶CaCl 2 concentration at 2.5 g/L,sodium alginate concentration at 1.5%,immobilization time at 2 h,mass ratio of carrier to enzyme at 40 000∶1.According to the orthogonal experiment,The optimal conditions were as follows∶CaCl 2 concentration at 2.5 g/L,sodium alginate concentration at 2%,immobilization time at 1 h,mass ratio of carrier to enzyme at 35 000∶1.In this condition,the recovery rate of immobilized enzyme was 75.43%.Compared with the free enzyme,the optimum catalytic temperature and pH of immobilized enzyme increased by 0℃and 0.5,and reached at 37.5℃and 7.5,respectively.Compared with free enzyme,Km and Vmax increased by 0.4 and 0.74,respectively.The immobilized enzyme maintained 50%of original enzyme activity after 6 times for continuous using.
作者
党湫菂
朱建全
寸聪瑞
冯璐
毕宜颖
赵蕤
谭艳
周嘉裕
廖海
DANG Qiu-di;ZHU Jian-quan;CUN Cong-rui;FENG Lu;BI Yi-ying;ZHAO Rui;TAN Yan;ZHOU Jia-yu;LIAO Hai(Southwest Jiaotong University,School of Life Science and Engineering,Chengdu 610031,China;Chengdu military district general hospital of the people's liberation army,Chengdu 610083,China)
出处
《天然产物研究与开发》
CAS
CSCD
北大核心
2018年第8期1294-1299,共6页
Natural Product Research and Development
基金
国家自然科学基金(31500276)
四川省应用基础研究项目(2017JY0222)
成都市科技技术研发项目(2015-HM01-00051-SF
2016-HM01-00260-SF)
中央高校基本科研业务费医学类专题研究项目(2682016YXZT09)
国家级大学生创新创业训练计划(201710613069)
西南交通大学本科教育教学研究与改革重点项目(1502024)
四川省重点研发项目(2018SZ0061)
