Screening UDP-Glycosyltransferases for Effectively Transforming Stevia Glycosides: Enzymatic Synthesis of Glucosylated Derivatives of Rubusoside.

Five plant-derived uridine diphosphate glycosyltransferases (UGTs) that catalyzed the glucosylation of stevia glycosides (SGs) were uncovered as the result of sequence mining considering the catalytic residues and conserved motifs of the known UGTs. Thereinto, Lb UGT from Lycium barbarum with high activity toward rubusoside has been enzymatically characterized. The recombinant Lb UGT was demonstrated to catalyze the β-1,6-glucosylation at C19 of rubusoside, producing a monoglucosyl derivative 13-[( O -β-d-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(6- O -β-d-glucopyranosyl-β-d-glucopyranosyl) ester], which was then submitted to a β-1,2-glucosylation by Lb UGT, resulting in a diglucosyl derivative 13-[( O -β-d-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(2- O -β-d-glucopyranosyl-6- O -β-d-glucopyranosyl-β-d-glucopyranosyl) ester]. The di-glycosylated product of rubusoside showed an obvious increase in sweetness intensity (134 times sweeter than 5% sucrose) and almost eliminated the unpleasant bitter taste. This work will provide a reference for the taste improvement of SGs.