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.

Platycodon grandifloras L.  /  glycosyltransferase  /  UDP-glucose  /  quercetin  /  functional verification  /  enzyme kinetics