UDP-糖基转移酶GmSGT2催化金盏花苷E的半乳糖基修饰  

Galactosylation of calunduloside E by UDP-glycosyltransferase GmSGT2from Glycine max

作　　者：孙秋艳 郭芳 李春 冯旭东 SUN Qiuyan;GUO Fang;LI Chun;FENG Xudong(Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering of the Ministry of Industry and Information Technology,Institute of Biochemical Engineering,Department of Chemical Engineering,School of Chemistry and Chemical Engineering,Beijing Institute of Technology,Beijing 100081,China;Key Laboratory for Industrial Biocatalysis of the Ministry of Education,Department of Chemical Engineering,Tsinghua University,Beijing 100084,China)

机构地区：[1]北京理工大学化学与化工学院化学工程系生物化工研究所医药分子科学与制剂工程工业和信息化部重点实验室,北京100081 [2]清华大学化学工程系工业生物催化教育部重点实验室,北京100084

出　　处：《生物加工过程》2024年第1期9-16,共8页Chinese Journal of Bioprocess Engineering

基　　金：国家重点研发计划(2019YFA0905700);北京市科技新星计划(Z191100001119099)。

摘　　要：通过qPCR从大豆cDNA文库克隆得到UDP-糖基转移酶GmSGT2的基因,构建工程菌株E.coli BL21/pET28a-GmSGT2,通过His-tag标签法得到纯化的GmSGT2,探究GmSGT2底物特异性,考察pH、温度对GmSGT2的影响,测定其催化金盏花苷E的动力学常数,通过分子对接阐明GmSGT2的底物识别机制。结果表明:GmSGT2可将1分子半乳糖转移到金盏花苷E糖上的C2位置,实现糖上加糖。除UDP-半乳糖外,GmSGT2还可识别UDP-葡萄糖、UDP-木糖和UDP-阿拉伯糖,相对酶活为24.67%~37.60%。GmSGT2催化金盏花苷E的最适温度为40℃,最适pH为7.5,催化效率k_(cat)/K_m为2.71 L/(mmol·s)。分子对接结果表明,His20是GmSGT2的催化氨基酸,Ser19、Ala146、Arg260、Tyr262、Ser291、Leu353、Trp370、Asn371、Thr372和Glu391参与识别底物。GmSGT2催化金盏花苷E半乳糖苷化的研究为酶法合成半乳糖苷衍生物提供指导意义。The construction of E.coli BL21/pET28a-GmSGT2 was achieved through the insertion of UDP-glycosyltransferase GmSGT2 gene,which was cloned from soybean cDNA through qPCR,into pET28a,and subsequent introduction into E.coli BL21(DE3).GmSGT2 enzyme,which was produced and purified according to His-tag method,was investigated in terms of its substrate specificity,optimal temperature and pH for enzymatic activity,as well as its Michaelis kinetics.Moreover,molecular docking was performed to elucidate the substrate recognition mechanism of GmSGT2 toward calunduloside E.As a result,GmSGT2 could specifically transfer one galactose moiety to C2 position of the glucuronic acid moiety of calunduloside E.In addition to UDP-galactose,GmSGT2 could also recognize UDP-glucose,UDP-xylose and UDP-arabinose with relative activities of 24.67%-37.60%.Regarding the GmSGT2-catalyzed galactosylation of calunduloside E,the optimum temperature and pH were 40℃and 7.5,respectively.The catalytic efficiency k cat/K m was 2.71 L/(mmol·s).The results from molecular docking showed His20 served as the catalytic amino acid,along with other residues including Ser19,Ala146,Arg260,Tyr262,Ser291,Leu353,Trp370,Asn371,Thr372 and Glu391 to recognize calunduloside E.Our research on GmSGT2-catalyzed galactosylation of calunduloside E will be beneficial for the enzymatic synthesis of other galactoside derivatives.

关 键 词：GmSGT2 UDP-糖基转移酶 糖基化 金盏花苷E UDP-半乳糖 分子对接 

分 类 号：Q55[生物学—生物化学]