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.