粉葛O-糖基转移酶PtUGT-6和PtUGT-7的功能表征及酶学特性研究OA

The dried root of Pueraria thomsonii,a plant of the Fabaceae family,features homology of medicine and food.It is rich in isoflavone glycosides such as puerarin and daidzin.Glycosylation,catalyzed by uridine diphosphate(UDP)glycosyltransferase(UGT),is a key post-modification step in isoflavone biosynthesis.However,the functional characterization of UGT in P.thomsonii remains limited.In this study,two putative O-glycosyltransferase genes,PtUGT-6 and PtUGT-7,were identified from the P.thomsonii transcriptome.Phylogenetic tree analysis revealed that PtUGT-6 and PtUGT-7 clustered closely with PlUGT15,an isoflavone 4',7-O-glycosyltransferase from P.lobata.In vitro enzymatic assays demonstrated that both PtUGT-6 and PtUGT-7 catalyzed the conversion of daidzein to daidzin.Enzymatic property analysis showed that the optimal reaction temperatures of PtUGT-6 and PtUGT-7 were 37 and 45℃,respectively,with an optimal pH of 9.0 for both enzymes.Furthermore,no metal ion dependence was exhibited.After 1 hour of the reaction,the substrate conversion rate reached approximately 80%and then tended to stabilize.Kinetic analysis revealed that the Km values of PtUGT-6 and PtUGT-7 were 22.43 and 29.39 μmol·L-1,respectively,with corresponding Kcat values of 2.16×10-3 and 2.17×10-3 s-1 and Kcat/Km of 96.30 and 73.83 L·mol-1·s-1,respectively.In summary,this study identified two glycosyltransferase genes,PtUGT-6 and PtUGT-7,from P.thomsonii,which can catalyze the glycosylation of daidzein to daidzin.These findings provide important insights into the biosynthetic pathway of characteristic isoflavone glycosides in P.thomsonii and offer a theoretical basis for improving the accumulation and quality of active compounds in this medicinal plant.