【作者】 富瑶瑶；

【导师】 金凤燮；

【作者基本信息】 延边大学 ， 有机化学， 2010， 博士

【摘要】 苷类是中草药的有效成分之一。但天然苷类物质并不是生理活性最佳的分子结构,皂苷口服后在体内经过肠道菌和消化系统酶的作用,转化为低糖基、次生苷被吸收而起药效。本文目的是利用生物酶法在体外将天然皂苷转化成易吸收、药效高的结构。为了得到高活性、易吸收的低糖基的柴胡皂苷及薯蓣皂苷,本文从微生物中获得了与传统糖苷酶性质不同的新型皂苷酶,并对新型柴胡皂苷酶和薯蓣皂苷酶进行了分离纯化、酶学性质、酶催化水解作用,及定向转化的研究。采用缓冲液抽提、硫铵沉淀、离子交换层析、凝胶电泳法从两种微生物Aspergillus oryzae c42和Absidia sp. d38中分别分离纯化了柴胡皂苷酶及薯蓣皂苷酶。柴胡皂苷酶被纯化了56倍,收率为1.4%；薯蓣皂苷酶被纯化了7.8倍,收率为3.6%。柴胡皂苷酶最适反应温度为40℃,最适pH为5.0；Cu++,Hg++,Ag+对酶活力有抑制作用,Ca++和Mg++对酶活力有激活作用。该酶在60℃以下、pH 4.0～7.0相对稳定。SDS-聚丙烯酰胺凝胶电泳测定该单亚基酶分子量约为58 kDa。薯蓣皂苷酶最适反应温度为40℃,最适pH为5.0；Cu++,Hg++对酶活力有抑制作用；Mg2+对酶活力有激活作用。该酶在50℃以下、pH 5.0～6.0相对稳定。SDS-聚丙烯酰胺凝胶电泳测定该单亚基酶分子量约为55 kDa。在柴胡皂苷酶的研究中发现,柴胡皂苷酶可以水解柴胡皂苷A(含有环氧醚键)和柴胡皂苷B2(不含环氧醚键)。首先将柴胡皂苷的3-O-β-D-(1→3)-Glc水解,形成3-O-β-D-Fuc-柴胡皂苷元,然后进一步水解3-O-β-D-Fuc-柴胡皂苷元的3-O-β-D-Fuc形成不带糖基的柴胡皂苷元。柴胡皂苷酶对糖基位置及苷元结构的选择性高,能较好的水解三萜类皂苷(柴胡皂苷),对糖基种类选择性低,可以水解3-C位的Glc、Fuc等不同种类糖基；对甾体类皂苷(薯蓣皂苷)不水解。在薯蓣皂苷酶的研究中发现,薯蓣皂苷酶首先将薯蓣皂苷的两个3-O-α-L-(1→4)-Rha和3-O-α-L-(1→2)-Rha水解,生成3-O-β-Glc-薯蓣皂苷元,然后进一步水解3-O-β-D-Glc-薯蓣皂苷元的3-O-β-D-Glc生成不带糖基的薯蓣皂苷元。薯蓣皂苷酶还能将3-O-[α-L-(1→4)-Ara,α-L-(1→2)-Rha]-β-D-Glc-薯蓣皂苷元的3-O-α-L-(1→2)-Rha水解,生成3-O-α-L-Ara-β-D-Glc-薯蓣皂苷元,然后进一步水解α-L-(1→4)-Ara生成3-O-β-D-Glc-薯蓣皂苷元,最后水解3-O-β-D-Glc生成不带糖基的薯蓣皂苷元。薯蓣皂苷酶仅对甾体类皂苷(薯蓣皂苷、3-O-[α-L-(1→4)-Ara,α-L-(1→2)-Rha]-β-D-Glc-薯蓣皂苷元)具有较好的水解作用,对三萜类皂苷(柴胡皂苷、白头翁皂苷及朱砂根皂苷)不具有水解作用。该酶糖基种类的选择性不高,可以水解Glc、Ara、Rha等不同种类糖基,但对糖基位置苷元种类具有较高的选择性,只能水解甾醇类皂苷。定向转化柴胡皂苷时,柴胡皂苷酶在底物浓度20 mg／mL,40℃,pH 5.0条件下,反应24h水解20g柴胡皂苷得粗产物23.78g。柴胡皂苷酶经60%回收后仍具有很高的酶活力,其转化率大于60%。酶解产物经硅胶柱层析法分离纯化后,回收率为79.55%,水解掉两个糖基的柴胡皂苷单体(酶解产物Ⅰ)得率为21.85%,水解掉一个糖基的柴胡皂苷单体(酶解产物Ⅱ)得率为4.45%。经HPLC测定纯度达90%左右。定向转化薯蓣皂苷时,薯蓣皂苷酶在底物浓度15 mg／mL,40℃,pH 5.0条件下,反应30h水解20g薯蓣皂苷得粗产物22.54g。薯蓣皂苷酶经60%回收后仍具有很高的酶活力,其转化率大于60%。酶解产物经硅胶柱层析法分离纯化后,回收率为91.8%,薯蓣皂苷元单体得率为20.85%,3-O-β-D-Glc-薯蓣皂苷元单体得率为39.8%；3-O-α-L-Rha-β-D-Glc-薯蓣皂苷元单体得率为12.85%。经HPLC测定纯度达90%左右。柴胡皂苷酶和薯蓣皂苷酶对糖基位置及苷元结构的选择性高,柴胡皂苷酶不水解薯蓣皂苷、薯蓣皂苷酶不水解柴胡皂苷；两种酶对糖基种类的选择性低、能够水解多种糖基；这不同于国际酶学委员会认定的“一种糖昔酶只能水解一种糖苷键”的规律；柴胡皂苷酶和薯蓣皂苷酶是新发现的特异酶。本文在证明酶法定向转化柴胡皂苷及薯蓣皂苷制备低糖基皂苷或苷元的可行性的同时,为深入研究糖苷酶由其是皂苷糖苷酶奠定了一定的实验基础。更多还原

【Abstract】 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.更多还原