【作者】 李翠翠；

【导师】 金凤燮；

【作者基本信息】 大连工业大学 ， 发酵工程， 2009， 硕士

【摘要】 本文主要研究的内容是以人参二醇类皂苷PPD为底物,利用微生物酶法转化生产人参稀有皂苷C-K,利用高压硅胶柱分离法提取人参皂苷C-K,并用高效液相色谱对其纯度进行确认。为了得到具有较高转化能力的皂苷糖苷酶的菌株,选取了4种菌株,比较产人参皂苷C-K酶的能力。实验发现sp.848产的生成人参皂苷C-K酶的能力最强。该菌株在28℃至30℃条件下发酵7天,产酶最好。真菌sp.848所产酶,经过DEAE-Cellulose离子交换层析柱分离后,再通过聚丙烯酰胺凝胶电泳提纯,得到纯酶,并进行酶的性质的研究。在SDS-聚丙烯酰胺凝胶电泳中其分子量约为74kDa；不同于前届研究生王亮从sp.48产酶的分子量77kDa。该酶最佳pn5.0,酶最佳反应温度是45℃。其纯酶对于人参二醇类皂苷Rb1、Rb2、Rc等单体降解能力强,说明该酶为多种糖基的水解酶,是属于人参皂苷糖苷酶Ⅰ型酶；该酶对于人参皂苷Rb1、Rb2、Rc可较大程度转化为人参皂苷C-K,对于人参皂苷Rd转化成C-K较弱。依据最适的酶反应条件扩大酶反应。酶反应产物利用D-296树脂脱色柱除去杂质,除去杂质占总重的4.74%。再经石油醚脱酯,脱酯共除去杂质占总重的7.46%。常压硅胶柱洗脱速度慢,高压硅胶柱洗脱快,利用高压硅胶柱层析法进行分离提纯,以V(氯仿)：V(甲醇)=9.5：0.5的混合液为洗脱剂进行洗脱,从335g粗品中得到138g纯度为80%-85%的人参皂苷C-K,其重量收率为41.2%。硅胶柱分离得到的人参皂苷C-K用重结晶方法精制,得率90%,高效液相色谱检测其纯度为97%。更多还原

【Abstract】 The purpose of this paper is to study the characterization of the ginsenosidase which can hydrolyze ginsenoside the protopanaxadiol type saponin (PPD) to ginsenoside compound K(C-K), the ginsenoside compound K(C-K) from enzyme reaction was isolated by silica gel column chromatography, the purity was examined by the method of HPLC.In order to get the strain producting the enzyme which could hydrolyse the PPD to the ginsenoside compound K(C-K) as much as possible, we selected4strains to study the ability of producting ginsenside-glycosidase, sp.3, sp.18, sp.48, sp.848. The experiment found sp.848yielded the highest ginsenside-glycosidase, Futhermore, it was shown that7days was the best period of enzyme fermentation.The enzyme isolated from sp.848was purified by a column of DEAE-Cellulose and polyacrylamide gel electrophoresis, and the characteration of enzyme were studied. After purified by a column of DEAE-Cellulose and molecular weight with the method of SDS-polyacrylamide gel electrophoresis, the molecular weight was74kDa different from the molecular weight77kDa studied by Wang Liang. The results showed the optimum pH was5.0, and the optimum temperature was at45℃. The enzyme which is ginsenoside glycosidase I can hydrolyse the ginsenoside Rb1, Rb2, Rc to the ginsenoside compound K(C-K) well, but can not hydrolyse the ginsenoside Rd well.Refering to the fittest condition we enlarged the enzyme reaction. The decolorizing by D-296removed4.74%impurity, and ungreasing esters by petroleum benzin was removed7.46%impurity. Finally, it was purified by the Silica gel column. The best mobile phase was the mixture of V(chloroform):V(methanol)=9.5:0.5. We get the ginsenoside C-K138g from crude ginsenoside C-K335g. In the silica gel column, ginsenoside C-K was obtained41.2%. After crystallizating, the pure C-K yield was90%. The ginsenoside C-K purity was97%by the method of HPLC.更多还原