【作者】 张春枝；

【导师】 安利佳； 金凤燮；

【作者基本信息】 大连理工大学 ， 生物化工， 2002， 博士

【摘要】 本文以专一水解人参皂苷特定糖基为目的，进行了人参皂苷糖基水解酶的分离纯化、性质研究、以及人参皂苷Rg3和Rc的定向酶解反应，证明了酶法定向改造人参皂苷结构的可能性。 采用缓冲液抽提、硫铵沉淀、离子交换层析法从人参根中分离纯化了两种人参皂苷糖基水解酶——人参皂苷-β-葡萄糖苷酶和人参皂苷-α-阿拉伯糖苷酶。人参皂苷-β-葡萄糖苷酶被纯化了16倍，收率为14.4％，人参皂苷-α-阿拉伯糖苷酶被纯化了18.5倍，收率为4.6％。PAGE检测纯化后的酶为单一谱带。 人参皂苷-α-葡萄糖苷酶能水解人参皂苷Rg3生成人参皂苷Rh₂，最适作用温度为55-60℃，最适pH为5.0，Ca⁺⁺对该酶有激活作用，Cu⁺⁺有抑制作用。该酶在60℃以下，pH4.0-7.0稳定，SDS-PAGE测得分子量为59kDa。此酶N-端氨基酸序列为SLDANYVPKYVTLPL，与已知序列的β-葡萄糖苷酶无同源性，人参皂苷-β-葡萄糖苷酶在水解特性上也不同于传统的β-葡萄糖苷酶（EC3．2．1．21）。本文还建立了人参皂苷-β-葡萄糖苷酶催化Rg3反应的米氏方程。 人参皂苷-α-阿拉伯糖苷酶能水解人参皂苷Rc生成人参皂苷Rd，最适作用温度为50℃，最适pH为5.0，Cu⁺⁺对酶有抑制作用。该酶在60℃以下，pH4.0-6.0稳定，SDS-PAGE测得分子量为86kDa。 人参皂苷糖基水解酶在底物浓度10mg／ml，pH5.0，55℃的条件下水解人参皂苷Rg3，反应24h，Rg3的转化率可达到60％。经饱和正丁醇萃取，硅胶柱分离，酶解产物Rh₂纯度可达90％，得率为32％。人参皂苷-β-葡萄糖苷酶能水解20（S）-Rg3和20（R）-Rg3分别生成20（S）-Rh₂和20（R）-Rh₂，水解速度大体相同。核磁共振(¹HNMR、¹³CNMR)和质谱（MS）检测结果显示，酶解20（S）-Rg3所得产物为20（S）-Rh₂，系统名称为3-O-（β-D-吡喃葡萄糖基）-达玛-24-烯-3β，12β，20（S）-三醇；酶解20（R）-Rg3所得产物为20（R）-Rh₂，系统名称为3-O-（β-D-吡喃葡萄糖基）-达玛-24-烯-3β，12β，20（R）-三醇，分子式为C₃₆H₆₂O₈，分子量为622。 人参皂苷糖基水解酶在底物浓度30mg／ml，pH5.0，50℃的条件下水解人参皂苷Rc，反应24h，Rc的转化率可达到60％。经饱和正丁醇萃取，硅胶柱分离，酶解产物Rd纯度可达90％，得率为34％。核磁共振(¹HNMR、¹³CNMR)和质谱（MS）检测结果表明，酶解产物为20（S）-人参皂苷Rd，系统名称为3-O-（β-D-吡喃葡萄糖基-（1→2）-β-D-吡喃葡萄糖基）-20-O-（β-D-吡喃葡萄糖基）-达玛-24- 摘 要烯与p，126，20o卜三醇，分子式为 C48H82Ol以 分子量为 946。 本文在证明酶法定向转化人参皂着制备稀有皂苦可行性的同时，为深入研究糖着水解酶尤其是皂苦糖基水解酶奠定了一定的实验基础。更多还原

【Abstract】 In order to hydrolyze the ginsenosides specifically, the ginsenoside- -glucosidase and the ginsenoside- -arabinofuranase are purified, characterized, and ginsenoside Rg3 and Re are hydrolyzed by the enzymes as well. It shows the possibility of hydrolyzing ginsenosides specifically. The ginsenoside-p-glucosidase and the ginsenoside-a-arabinofuranase from ginseng root are purified to one spot in PAGE by buffer extraction, (NH4)2SO4 precipitation and ion exchange chromatography.The ginsenoside-p-glucosidase hydrolyzes the ginsenoside Rg3 sugar moiety to ginsenoside Rh2. The optimal temperature is 55-60 , and the optimal pH is 5.0. Ca2+ ion has the positive effect, while Cu2+ has the negative effect on it. Its molecular weight is about 59 kDa in SDS polyacrylamide gel electrophoresis. The N-end sequence of ginsenoside-p-glucosidase is SLDANYVPKYVTLPL, which has no homology with the p-glucosidases whose protein sequence are known. The hydrolysis of ginsenoside-p-glucosidase is different from the original exocellulase such as -glucosidase (ECS.2.1.21). In addition, the Michaelis-Menten model of ginsenoside- -glucosidase is built.The ginsenoside-a-arabinofuranase hydrolyzes the ginsenoside Re to ginsenoside Rd. The optimal temperature is 50 , and the optimal pH is 5.0. Cu2+ inhibites the enzyme activity. Its molecular weight is about 86 kDa in SDS polyacrylamide gel electrophoresis.In hydrolyzing ginsenoside Rg3 to Rh2 by the saponin glycosidases, the conversion rate is 60% in the condition of substrate Rg310mg/ml, pH5.0, 55 , 24h. After butanol extraction and separation on the silica gel column, the purity of the product Rh2 is about 90% and the yield is 32%. The ginsenoside-p-glucosidase can hydrolyze both 20(S)- Rg3 and 20(R)- Rg3 to form 20(S)- ginsenoside Rh2, i. e. 3-O-(p-glucopyranosyl)-dammar-24-en-3p, 12P, 20(S)-triol and 20(R)- ginsenoside Rh2, i. e. 3-O-(p-glucopyranosyl)-dammar-24-en-3p, 12p, 20(S)-triol respectively, according to the data of the ’HNMR, 13CNMR and MS. The reaction rate of 20(S)-Rg3 and 20(R)- Rgs is nearly the same.In hydrolyzing ginsenoside Re to Rd by the ginsenoside-cc-arabinofuranase, the conversion rate is 60% in the condition of substrate Re 30mg/ml, pH5.0, 50 , 24h. After butanol extraction and separation on the silica gel column, the purity of the product Rd is about 90% and the yield is 34%. The product from Re is 20(S)-Rd, i. e. 3-O-(p-glucopyranosyl-(1 - 2)-p-lucopyranosyl)-20-O-(p-glucopyranosyl)- dammar 24-en-3p, 12(3, 20(S)-triol according to the data of the ’HNMR, 13CNMR and MS.All this proves the possibility of transforming ginsenosides into the rare ginsenosides. It also lays a foundation on studying glycosidase especially saponin glycosidase further.更多还原