液相色谱-质谱联用-定向进化酶组合技术应用于三萜类皂苷转化研究  被引量:1

Liquid Chromatography-Mass Spectrometry-Directed Evolution Enzyme Combination Technique for Investigation of Triterpenoid Saponin Transformation

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作  者:孙红梅 陆泽林 赵幻希 郑飞 李晶 刘明 越皓 于珊珊 SUN Hong-Mei;LU Ze-Lin;ZHAO Huan-Xi;ZHENG Fei;LI Jing;LIU Ming;YUE Hao;YU Shan-Shan(Jilin Ginseng Academy,Changchun University of Chinese Medicine,Changchun 130117,China)

机构地区:[1]长春中医药大学吉林省人参科学研究院

出  处:《分析化学》2020年第1期129-136,共8页Chinese Journal of Analytical Chemistry

基  金:吉林省科技发展计划项目(No.20170307029YY);吉林省教育厅"十三五"科学技术项目(No.JJKH20181235KJ)资助~~

摘  要:利用定向进化技术重组糖苷酶ABQ,转化不同结构的三萜类皂苷(商陆皂苷甲、三七皂苷R 1和柴胡皂苷A),通过高分离度液相色谱-四极杆飞行时间质谱鉴定转化的产物结构,阐明其转化机制。结果表明,重组糖苷酶ABQ能够催化商陆皂苷甲、三七皂苷R 1和柴胡皂苷A侧链糖苷键的水解,转化为商陆皂苷乙、三七皂苷R 2、柴胡次皂苷F和柴胡皂苷元F。根据色谱-质谱对反应产物的实时分析,ABQ对这3种三萜皂苷表现出不同的底物选择性,重组糖苷酶ABQ能够催化商陆皂苷甲C-3位外侧的葡萄糖水解,生成商陆皂苷乙;三七皂苷R 1的C-20位葡萄糖被水解,生成三七皂苷R 2;重组糖苷酶ABQ能够首先催化柴胡皂苷A的C-3位葡萄糖水解,生成柴胡次皂苷F,并继续催化C-3位的岩藻糖苷水解,生成柴胡皂苷元F。定向进化技术重组酶能够提高酶活力和作用环境适应能力,色谱-质谱联用通过原型和产物实时检测评价重组酶的作用。The directed evolution technology was used for recombination of glycosidase ABQ to transform triterpenoid saponins with different structures,including Esculentoside A,Notoginsenoside R 1 and Saikosaponin A,and the biotransformation process of the triterpenoid saponins was investigated by rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry(RRLC/Q-TOF-MS).The result showed that ABQ had high activities for Esculentoside A,Notoginsenoside R 1,Saikosaponin A,however,it showed different substrates selectivity for the three saponins.Besides,ABQ could catalyze the hydrolysis of C-3 lateral glucose of Esculentoside A and the C-20 lateral glucose of Notoginsenoside R 1 to produce Esculentoside B and Notoginsenoside R 2,repectively.And the recombinant glycosidase ABQ could catalyze the hydrolysis of C-3 lateral glucose of Saikosaponin A to produce Prosaikogenin F and then further catalyzed the hydrolysis of C-3 fucoside to produce Saikogenin F.In brief,the directed evolution technology could improve enzyme activity and environmental adaptability,and the chromatography-mass spectrometry could be used to evaluate the role of recombinant enzymes by real-time detection of prototypes and products.

关 键 词:三萜皂苷 生物转化 液相色谱-质谱联用 酶定向进化技术 

分 类 号:R28[医药卫生—中药学]

 

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