Catalytic Hydrolysis Of Pulchinenoside-glycosidase

To study on the enzyme reaction of Pulsatilla chinensis saponin monomer, strain of enzyme production was opted and enzyme cultivate conditions were optimized. Then substrates and enzymatic products were seperated. The molecular weights of glycosidase, enzyme reaction conditions and enzyme characters were analysed.Saponin was extracted from Pulsatilla chinensis by alcohol, total saponin was purified by AB-8and D-296macroporous resin. Four kinds of substrate monomer were obtained by the proportion in8.5:1.5and8.3:1.7chloroform-methanol as mobile phase. The yields were10.6%,13.8%,8.20%,15.3%, and the purity of substrates were83.94%,81.99%,80.18%,83.40%, respectively.The pulchinenoside-glycosidase was produced by the Absidia sp.P00r, obtained27.9g enzymatic products, and the yield was69.75%. Three kinds of enzymatic product monomer were obtained by the proportion in8.5:1.5chloroform-methanol as mobile phase. The yields were15.0%,7.10%,10.3%, and the purity of products were93.45%,91.28%,99.90%, respectively.Pulchinenoside-glycosidase was seperated and purified by DEAE-cellulose52column and sephadex G-100column, obtained two kinds of glycosidase. Their molecular weights were approximately determined as73.1kDa,58.8kDa.The optimum conditions of enzyme I were:pH5.0, the range of stabilization is pH4.0-7.0; temperature40℃, stable below50℃. It was not effected by K+, Na+, Mg2+, Ca2+, the activity was promoted by low concentration of K+, Na+, the Cu2+, Fe3+and high concentration of Zn2+showed inhibitory effect. For enzyme I, the Vmax is0.386mmol/(L-min), the Km is17.44mmol/L.The optimum condition of enzyme II were:pH5.0, the range of stabilization is pH4.0-7.0; temperature40℃, stable below50℃; It was not effected by Na+, K+, Mg2+, the activity was promoted by low concentration of Mg2+, Na+, the Cu2+, Fe3+, Zn2+and high concentration of Ca2+showed inhibitory effect. For enzyme II, the Vmax is19.23mmol/(L-h),the Km is15.67mmol/L.