目的 以桔梗Platycodon grandiflorus中糖基转移酶为研究对象，进行糖基转移酶PgUGT72B21基因的生物信息学分析、基因克隆与重组质粒构建、蛋白表达与纯化、催化功能验证、酶学性质研究、底物宽泛性考察。方法 基于桔梗转录组数据筛选得到桔梗中关键糖基转移酶PgUGT72B21，进行基因克隆；利用基因重组技术构建了原核表达载体pET-28a-PgUGT72B21，转化至大肠杆菌BL21（DE3）感受态；根据His标签蛋白纯化试剂盒说明进行蛋白纯化，应用SDS-PAGE凝胶电泳检测蛋白的表达情况；通过HPLC和LC-MS系统检测酶促反应产物。结果 通过克隆得到的桔梗糖基转移酶基因PgUGT72B21开放阅读框（open reading frame,ORF）长度1 407 bp，编码468个氨基酸残基，相对分子质量约为51 000,C末端存在高度保守的植物次生产物糖基转移酶（plant secondary product glycosyltransferase,PSPG）基序。系统进化分析表明，该糖基转移酶属于UGT72家族。体外酶促结果显示，PgUGT72B21可催化槲皮素C3位羟基糖基化生成异槲皮苷。通过异源表达与纯化得到重组蛋白后对其酶学性质进行了分析，表明PgUGT72B21催化反应的最适pH为6.0，最适温度为60℃，反应2 h后的底物转化率达到最高。其催化槲皮素的酶动力学参数米氏常数（K_m）为390.10μmol/L，转化数（kcat）为11.10/min。进一步的底物宽泛性研究显示PgUGT72B21不仅能够催化槲皮素等黄酮醇类化合物，还能够催化芹菜素等黄酮类化合物。结论 发现的新糖基转移酶PgUGT72B21对于丰富糖基化工具酶库具有重要意义，且能为进一步解析桔梗中黄酮糖苷的糖基化过程奠定基础。

Objective Taking the glycosyltransferase in Platycodon grandiflorus as the research object, the bioinformatics analysis, gene cloning and recombinant plasmid construction, protein expression and purification, catalytic function validation, enzymatic properties, and substrate promiscuity of the glycosyltransferase gene PgUGT72B21 were investigated. Methods The key glycosyltransferase PgUGT72B21 was screened from the transcriptome data of P. grandiflorus and subsequently cloned. A prokaryotic expression vector, pET-28a-PgUGT72B21, was constructed using gene recombination technology and transformed into Escherichia coli BL21（DE3） competent cells. Protein purification was performed according to the instructions of the His-tagged protein purification kit, and protein expression was detected by SDS-PAGE electrophoresis. The enzymatic reaction products were analyzed using HPLC and LCMS systems. Results P. grandiflorus glycosyltransferase gene Pg UGT72B21 obtained through cloning had an open reading frame（ORF） of 1 407 bp, encoding 468 amino acid residues with a relative molecular mass of approximately 51 000. A highly conserved plant secondary product glycosyltransferase（PSPG） motif was identified at the C-terminus. Phylogenetic analysis indicated that this glycosyltransferase belongs to the UGT72 family. In vitro enzymatic assays demonstrated that PgUGT72B21 could catalyze the glycosylation of the C3 hydroxyl group of quercetin to produce isoquercitrin. After heterologous expression and purification, the recombinant protein was obtained, and its enzymatic properties were analyzed. The optimal pH and temperature for the catalytic reaction were determined to be 6.0 and 60 ℃, respectively, with the highest substrate conversion rate achieved after 2 h of reaction. The kinetic parameters for quercetin catalysis were K_m = 390.10 μmol/L and kcat = 11.10/min. Further substrate promiscuity studies revealed that PgUGT72B21 could not only catalyze flavonols such as quercetin but also flavonoids such as apigenin. Conclusion The newly discovered glycosyltransferase PgUGT72B21 holds significant importance for enriching the glycosylation tool enzyme library and provides a foundation for further elucidating the glycosylation process of flavonoid glycosides in P. grandiflorus.

R286.12

中国中医科学院科技创新工程项目(CI2023E002);中国中医科学院中药研究所自主创新课题(ZXKT22048);国家自然科学基金项目(32200308);山西省基础研究计划(自由探索类)面上项目(202403021211036)