生物加工过程2024，Vol.22Issue(1)：9-16,8.DOI:10.3969/j.issn.1672-3678.2024.01.002
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 GmSGT2 from Glycine max
摘要
Abstract
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 kcat/Km 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Key words
GmSGT2/UDP-glycosyltransferase/glycosylation/calunduloside E/UDP-galactose/molecular docking分类
生物科学引用本文复制引用
孙秋艳,郭芳,李春,冯旭东..UDP-糖基转移酶GmSGT2催化金盏花苷E的半乳糖基修饰[J].生物加工过程,2024,22(1):9-16,8.基金项目
国家重点研发计划(2019YFA0905700) （2019YFA0905700）
北京市科技新星计划(2191100001119099) （2191100001119099）
