Studies On Property And Application Of Endothelium Corneum Glucosidase

In this paper, Endothelium Coneum glucosidase was extracted from Chinese traditional medicine to hydrolyze soybean isoflavones. It converted combined soybean isoflavone glycosides into the corresponding aglycones which had much higher bio-activity. Crude enzymes were gained through distillation, ammonium sulfate precipitation, and re-dissolution. After glucosidase separated by separation medium, the enzyme qualities were studied. Then it was used to hydrolyze soybean isoflavones and the hydrolysis process was optimized.DEAE-cellulose DE-52was choosed as the separation medium. By SDS-PAGE, The glucosidase’s weight was confirmed. Comparing with hydrolysis of soybean isoflavone isomers, icariin and rutin, the result was presumed Endothelium Coneum glucosidase could hydrolyze some single glucoside bond.The crude enzymes of Endothelium Coneum were separated into five components by DEAE-cellulose DE-52under the conditions of loading amount:3mL, elution speed:1mL/min, pH:7.0ammonium sulfate buffer used as eluant, sodium chloride grads:0-0.2M. TLC results showed soybean isoflavones were transformed by the peak5.The molecular weight of Endothelium Coneum glucosidase measured by SDS-PAGE was66kDa. After the single and orthogonal experiments, the optimal hydrolysis conditions were pH5at35℃for15hours with the3500U/mL addition of enzyme quantity and4mg/mL substrate concentration. Through chromatogram, the hydrolysis rate reached up to99%under this condition.Soybean isoflavone mixture were separated into different monomer by silica colum. Three soybean isoflavone aglycones (daidzein, genistein, glycitein) were separated with dichloromethane and ethyl acetate as eluant. Four soybean isoflavone glucosides were gained with dichloromethane and dichloromethane:ethyl acetate=5:1as eluant. The purification rate of separations could reach up to above90%. Hydrolysis actions were act on these separated soybean isoflavones which hydrolysis rate were all above95%.In conclusion, Endothelium Corneum glucosidase which could convert soybean isoflavone glucosides into the corresponding aglycones which had higher bio-activity. Besides, Comparing with hydrolysis of icariin and rutin, it could be concluded that Endothelium Coneum glucosidase could hydrolyze some single glucoside bond.