Purification And Characteristics Of Uridine 5'-diphosphoglucose (UDPG) : Ginsenoside Rh₂ Glucosyltransferase (UGRh₂GT)

Glycosyltransferases are ubiquitous in plant cells. Glycosyltransferases link actived sugar residues to small molecule acceptor substrates via glycosidic bond in the course of plant secondary metabolites generated. The study of ginsenoside Rd glucosyltransferase of Panxa notoginseng was reported, while other ginsenoside glycosyltransferases in ginsengs were not reported, therefore, the study of ginsenoside Rh2 glucosyltransferase was innovative. Glucosyltransferases play an important role in ginsenoside biosynthesis, this work to study ginsenoside Rh2 glucosyltransferase in Panax ginseng cultured cells will offer information to elucidate the biosynthesis of ginsenoside Rg3, and be helpful to regulation of production of ginsenoside Rg3.In this experiment, the calli were induced from P. ginseng embryos and suspension cultures of the calli were established. Suspension cells of P. ginseng were used as the material for ginsenoside glucosyltransferase study. The activity of uridine 5'-diphosphoglucose (UDPG): ginsenoside Rh2 glucosyltransferase (UGRh2GT) was tried by UDPG as the glucose donor, ginsenoside Rh2 as substrate, the extract from P. ginseng suspension cells as an enzyme. The reaction products were determined by high performance liquid chromatography (HPLC). The temperature and pH used in determination of the enzyme activity were optimized by quadratic saturation D-optimum regression design. UGRh2GT was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography and ion exchange chromatography. The purity of the purified UGRh2GT was detected and the molecular weight of UGRh2GT was estimated by SDS-PAGE. The Km and Vmax of UGRh2GT with ginsenoside Rh2 were got by the Lineweaver-Burk method. EDTA, NH4+ and common metal ions were tested whether they influenced the activity of UGRh2GT; The activity of UGRh2GT was detected while ginsenoside Rd and aglycone protopanaxadiol was used as a substrate, separately.The activity of UGRh2GT was found in the extract from P. ginseng suspension cells. Optimum temperature and pH value of activity of UGRh2GT were 34.1℃and 9.3. UGRh2GT from the extract was purified 9.6-fold by hydrophobic chromatography. The part of UGRh2GT was collected for ion exchange chromatography. UGRh2GT was purified 43.7-fold by ion exchange chromatography. A single protein band of purified UGRh2GT was observed that molecular weight was 55kDa by SDS-PAGE. The Km value and Vmax of UGRh2GT were 0.2mM and 3.33nkat/mg, separately. The activity of UGRh2GT was promoted by Na +, K +, Li +, NH4 +, EDTA2+, Co2+, Ba2+, Ca2+, Mg2+ and Mn2+ , in which Li+ was the best. The activity of UGRh2GT was inhibited by Ag+, Fe2+, Zn2+, Hg2+ and Cu2+, Zn2+ and Hg2+, in which Zn2+ and Hg2+ were the strongest. UGRh2GT catalyzed neither ginsenoside Rd into Rb1, nor aglycone protopanaxadiol into ginsenoside Rh2 or Rg3, this suggested that UGRh2GT has its own acceptor or linkage specificity.