Biochemical characterization of UDP-glycosyltransferase UGT73C21 from Barbarea vulgaris for efficient conversion of protopanaxadiol to ginsenoside Rh2.

Ginsenoside Rh2, a potential anticancer agent originally isolated from the medicinal plant Panax ginseng, has low natural abundance and is difficult to extract, making biosynthetic production a promising alternative. UDP-glycosyltransferases (UGTs) that specifically catalyze protopanaxadiol (PPD) glycosylation to produce Rh2 have been widely identified in plants and microorganisms. In this study, we report that Barbarea vulgaris UGT73C21 effectively catalyzes this reaction. The enzyme was expressed in Escherichia coli and purified to electrophoretic homogeneity via Ni2⁺-affinity chromatography. It exhibited optimal activity at pH 8.0 (100 mM HEPES) and 45 °C. Mn2⁺, Mg2⁺, and Ca2⁺ significantly enhanced enzyme activity, whereas other tested metal ions reduced it. The kinetic parameters Km, Vmax, kcat, and kcat/Km were determined as 231.92 μM, 5.68 μM min-1, 0.053 s-1, and 228 M-1 s-1, respectively. In a reaction containing 0.2 mg/mL UGT73C21, 1 mM PPD, and 5 mM UDP-Glucose in HEPES buffer (pH 8.0), 0.55 mM Rh2 was produced within 1 h, corresponding to a 55% conversion rate. These results demonstrate that B. vulgaris UGT73C21 is an efficient biocatalyst for ginsenoside Rh2 biosynthesis.