茶树CCoAOMT原核表达及酶促条件优化  

Prokaryotic expression of CCoAOMT from Camellia sinensis and optimization of enzymatic conditions

作　　者：唐倩 陈丝 欧淑琼[1,2,4] 李勤 李娟 王坤波[1,2,3,4] TANG Qian;CHEN Si;OU Shuqiong;LI Qin;LI Juan;WANG Kunbo(National Research Center of Engineering and Technology for Utilization of Botanical Functional Ingredients,Changsha,Hunan 410128,China;Key Laboratory of Education Ministry for Tea Science,Changsha,Hunan 410128,China;Co-Innovation Center of Education Ministry for Utilization of Botanical Functional Ingredients,Changsha,Hunan 410128,China;Horticulture College,Hunan Agricultural University,Changsha,Hunan 410128,China)

机构地区：[1]国家植物功能成分利用工程技术研究中心,湖南长沙410128 [2]茶学教育部重点实验室,湖南长沙410128 [3]植物功能成分利用省部共建协同创新中心,湖南长沙410128 [4]湖南农业大学园艺学院,湖南长沙410128

出　　处：《湖南农业大学学报（自然科学版）》2020年第5期533-537,共5页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家科学技术部重点研发计划课题(2018YFC1604403);湖南省科学技术厅中央引导地方科技发展项目(2019XF5041);湖南省科学技术厅重点研发计划项目(2018NK2031)。

摘　　要：优化了重组茶树咖啡酰辅酶A–O–甲基转移酶基因(CCoAOMT)在大肠杆菌中的表达,采用Ni–NTA亲和层析柱对重组蛋白进行纯化。以表没食子儿茶素没食子酸酯(EGCG)为底物,进行体外酶促反应,采用HPLC测定反应产物EGCG3"Me的生成量。结果表明,CCoAOMT在E.coliBL21中能够高效表达,最优条件为诱导温度37℃,诱导时间4 h,异丙基–β–D–硫代半乳糖苷(IPTG)浓度0.5 mmol/L;重组蛋白经Ni–NTA亲和层析柱梯度洗脱达到了较好的纯化效果;重组酶在体外能够催化EGCG,合成EGCG3"Me,底物EGCG最佳浓度为0.05 mmol/L,最适温度为37℃,最适pH值为4。The expression of the recombinant caffeinyl-coenzyme A O-methyltransferase gene(CCoAOMT)in E.coli cells was optimized and the enzyme was purified by Ni-NTA affinity chromatography.The enzymatic reaction was carried out in vitro with epigallocatechin gallate(EGCG)as substrate,and EGCG3"Me,the reaction products,were analyzed by HPLC.As a result,CCoAOMT was highly expressed in E.coli BL21 and the best induction conditions of recombinant CCoAOMT was 0.5 mmol/L IPTG at 37℃for 4 h.The recombinant protein was purified by Ni-NTA affinity chromatography.The recombinant CCoAOMT could catalyze EGCG to EGCG3"Me in vitro and the optimal reaction conditions include substrate EGCG of 0.05 mmol/L,pH of 4 and temperature of 37℃.

关 键 词：茶树 咖啡酰辅酶A–O–甲基转移酶 原核表达 纯化 酶促合成 

分 类 号：S571.101[农业科学—茶叶生产加工]