3.5 Production of ginsenoside Rh2 by the SuSy-GT reaction
Ginsenoside Rh2, which was an important triterpene saponin and originally isolated from red ginseng, has diverse pharmacological activities, including anti-oxidation, hepatoprotection, anti-diabetes and anti-tumor.[34, 35] The engineered glucosyltransferase UGT51 (UGT51m) from Saccharomyces cerevisiae , with a seven-residue mutation (S801A/L802A/V804A/K812A/E816K/S849A/N892D), was previously reported that can efficiently transfer a glucosyl moiety onto the C-3-OH of PPD to produce the ginsenoside Rh2.[21] UDPG was used as the sugar donor for the glycosylation of PPD by UGT51m. In the present study, UGT51m and Mc SuSy prepared from E. coliBL21 (pRSF-UGT51m-Mc SuSym) were coupled to form a SuSy-GT system. The Arabidopsis -derived At SuSy1 which was widely applied in various glycosylation reactions, was used in the control reactions. The cascade reactions were carried out at pH 7.0 and 37 °C. Samples were taken at 0, 0.5, and 3 h, and the concentration of Rh2 was detected by HPLC. The production of Rh2 was raised with the increase of PPD concentration and accumulated rapidly in 30 min, then grew slowly later. When the initial concentration of PPD was 6 mM, the yield of Rh2 was about 76% in each system, which was the same as that of 8 mM PPD concentration, but the product was 1.3 times lower than that of 8 mM PPD (Fig. 4). By measuring the SuSy activity in both systems, the specific activity of Mc SuSym (53.2 mU/mg) was comparable to that ofAt SuSy1 (55.8 mU/mg). Results showed that Mc SuSym worked as well as At SuSy1 for UDPG regeneration. As a result, 6.02 mM (3.75 g/L) of Rh2 was synthesized from 8 mM PPD byMc SuSym-UGT51m.