Two Type Of Special Saponin-Glycosidases Hydrolyzing Saikosaponin And Dioscin

Saponin is one of the physiologically active ingredients in Chinese traditional medicine, but saponin in its natural form cannot be directly absorbed and utilized by human body. After oral intake, the glycosides of saponins are hydrolyzed by digestive enzymes and/or intestinal bacteria into low-sugar-saponin and aglycone, which are absorbed slowly in gastrointestinal tract to exhibit physiological activity. Therefore, modification of natural saponins by enzymes in vitro to produce more active second saponins that can be easily absorbed and utilized by human body would add great value to functional foods and medicines made from herbs. In this paper, saponin-glycosidase, a new type of glycosidase, which could hydrolyze saikosaponin and dioscin to low-sugar-saponin were studied. In order to hydrolyze the saikosaponin and dioscin specifically, the saikosaponin-glycosidase and dioscin-glycosidase were isolated, purified, and characterized.These two enzymes cultured from Aspergillus oryzae c42 and Absidia sp. d38 strain separately are purified to one sport in PAGE after buffer extraction, (NH4)2SO4 precipitation and ion exchange chromatography. In the purification, the saikosaponin-glycosidase yield was 1.4% and the enzyme specific activity was increased by 56 times; the dioscin-glycosidase yield was 3.6% and the enzyme specific activity was increased by 7.8 times. HPLC(C8) was used to further check the purity of these two glycosidases. Only one peak appeared on HPLC, respectively, indicating that the two glycosidases separated by DEAE and PAGE were already pure enzyme.The optimum temperature and pH of the saikosaponin-glycosidase was 40℃and 5.0, respectively:and stable under 60℃, pH 4.0～7.0. The activity of saikosaponin-glycosidase was not apparently affected by the Na+ and K+ ions, but significantly inhibited by the Cu++, Hg++ and Ag+ions; and slightly affected by the Ca++ and Mg++ ions. The molecular weight of saikosaponin-glycosidases was about 58 kDa in the SDS-polyacrylamide gel electrophoresis.The optimal temperature of dioscin-glycosidase was 40℃; the optimal pH was.5.0; and stable under 50℃, pH 5.0～6.0. The activity of dioscin-glycosidase was not affected by the Na+, K+and Mg2+ions; it was significantly inhibited by the Cu2+and Hg2+ions; and it was slightly affected by the Ca2+ ions. The molecular weight of dioscin-glycosidases was about 55 kDa in the SDS-polyacrylamide gel electrophoresis.The saikosaponin-glycosidase not only hydrolyzed 3-O-β-D-(1→3)-glucoside of saikosaponin A (containing 13β,28-epoxy ether bond) into 3-O-β-D-Fuc-saikosapogenin A, further hydrolyzed 3-O-β-D-Fuc-saikosapogenin A into saikosapogenin A; but also hydrolyzed 3-O-β-D-(1→3)-glucoside of saikosaponin B2 (non-containing 13β,28-epoxy ether bond) into 3-O-β-D-Fuc-saikosapogenin B2; further hydrolyzed 3-O-β-D-Fuc-saikosapogenin B2 into saikosapogenin B2. The saikosaponin-glycosidase hydrolyzed the Glc,Fuc of triterpenoide saponins in 3-C, but not hydrolyzed steroidal saponins.The dioscin-glycosidase gradually hydrolyzes 3-O-α-L-Rha of dioscin to 3-O-α-L-Rha-β-D-Glc-diosgenin; further rapidly hydrolyzes the other a-L-Rha to main intermediate products 3-O-β-D-Glc-diosgenin; and subsequently hydrolyzes intermediate products to final product of aglycone. the enzyme also gradually hydrolyzes 3-O-α-L-(1→4)-Ara,3-O-α-L-(1→2)-Rha.andβ-D-Glc of [3-O-α-L-(1→4)-Ara, 3-O-α-L-(1→2)-Rha]-β-D-Glc-diosgenin into final product diosgenin. The dioscin-glycosidase hydrolyzed the Glc,Ara,Rha of steroidal saponins, but not hydrolyzed triterpenoide saponins.20g saikosaponin was hydrolyzed by saikosaponin-glycosidase under the conditions of that substrate concentration 20 mg/mL,40℃, pH 5.0 and 24h, obtaining 23.78g crude product. The enzyme activity was still high after recycle in 60%. After separation on the silica gel column, the yield of saikosaponin enzymatic productⅠandⅡwere 21.85% and 4.45%, respectively. And the purity of both enzymatic products was 90% detecting by HPLC.20g dioscin was hydrolyzed by dioscin-glycosidase under the conditions of that substrate concentration 15 mg/mL,40℃, pH 5.0 and 30h, obtaining 22.54g crude product. The enzyme activity was still high after recycle in 60%. After separation on the silica gel column, the yield of diosgenin was 20.85%, the yield of 3-O-β-D-Glc-diosgenin was 39.8%, and the yield of 3-O-α-L-Rha-β-D-Glc-diosgenin was 12.85%. The purity of these enzymatic products was 90% detecting by HPLC.The multi-glycoside nature of saikosaponin-glycosidase and dioscin-glycosidase significantly differs from what is considered norm for glycosidases described in Enzyme Nomenclature by NC-IUBMB "one enzyme hydrolyzes one type of glycoside",indicating that saikosaponin-glycosidase and dioscin-glycosidase were new special enzyme.All this proved the possibility of transforming saikosaponin and dioscin into the low-sugar glycoside saponins. It also provided a foundation on studying glycosidase especially saponin glycosidase further.