P-22 ヨウシュヤマゴボウ培養細胞による配糖化と糖転移酵素の結晶構造解析

The biotransformation of the foreign substrate using the living cells is interested in medical, pharmaceutical and organic synthesis fields. In such a status we had studied the biotransformation of foreign substrate using plant cultured cells and it was found that the plant cells have the conversion ability such as stereoselective reduction, enantioselective oxidation, regioselective hydroxylation and glycosylation. Saponine is accumulated in plants as secondary metabolite, and it’s ability draws an international attention as a functional material. Also glycosylation was useful primarily to enhance water-solubility and thermal stability of compounds. We had investigated the production of saponines in one step and at reasonably low cost by using plant cultured cells. Recently we extract glucose transferase (PaGT2 and PaGT3) derived from plant cells. To synthesize the high functional saponines using PaGT2 and PaGT3, we studied the biotransformation of substrate using PaGT2 and PaGT3 enzyme. To produce the glycoconjugates efficiently, we used the purified enzyme, and the recombinant E.coli cells containing the PaGT2 and PaGT3 gene as biocatalysts. We used trans-resveratrol, pterostilbene, and piceatannnol as substrates. UDP-glucose: PaGT2 and PaGT3 activity was measured using stilbene and UDP-glucose as substrates. A standard reaction mixture consisted of 50 μM stilbene, 100 μM UDP-glucose, 5 mL of 50 mM potassium phosphate buffer (pH 7.2), and an enzyme. The mixture without an enzyme was preincubated at 30 °C, and the reaction was started by the addition of an enzyme. After incubation at 30 °C for 1 h, the reaction was stopped by the addition of trifluoroacetic acid. The reaction products were analyzed using a HPLC system. The glucosyl-acceptor specificity of the purified PaGT was examined with a variety of plant phenols (trans-resveratrol, pterostilbene, piceatannol, quercetin, kaempferol, and capsaicin) using UDP-glucose as the glycosyl donor. The phenols (trans-resveratrol, pterostilbene, piceatannol, quercetin, kaempferol, and capsaicin) were all inert as substrates for these enzymes. The enzymes, PaGTs, were highly specific for these phenol compounds. In this study, the X-ray crystal structure of PaGT2 complexed with resveratrol was also examined.