毁灭柱孢菌中一种人参皂苷Rb_1水解酶的纯化及酶学性质研究

Purification and Enzymatic Characters of a Ginseng Saponin Rb_1 Hydrolytic Enzyme from Cylindrocarpon destructans

通过DEAE-纤维素阴离子交换层析、30%～80%(NH4)2SO4盐析、Sepharose CL-6B凝胶过滤层析和Mono Q HR 5/5阴离子交换层析,从毁灭柱孢菌培养液中部分纯化出一种能够水解人参皂苷Rb1的β-葡萄糖苷酶F-I。F-I具有较好的pH稳定性和热稳定性,在pH 4.0～11.0范围内和55℃以下表现出良好的β-葡萄糖苷酶活性,其最适pH为5.0,最适温度为55℃。EDTA、Cu2+和Zn2+对该酶活性有较强的抑制作用。底物专一性分析表明,F-I能高特异性水解人工合成的底物pNPG,还能水解β-葡萄糖苷键连接的二糖如纤维二糖和龙胆二糖,说明此酶为一种β-葡萄糖苷酶。F-I对人参皂苷Rb1表现了较强的水解活性,而对人参皂苷Rb2和Rc的水解活性较低。该酶水解人参皂苷Rb1的路径为Rb1→Rd→F2→C-K。F-I对人参皂苷Rb1的这种高效水解为稀有人参皂苷的工业制备奠定了基础。

A ginseng saponin （GS） Rbl-hydrolytic enzyme β-glueosidase （F-I） was partially purified from cultural broth of the phylopathogenic fungus Cylindrocarpon destructans by DEAE-cellnlose anion exchange chromatography, 30% -80% （NH4）2SO4 salting-out, Sepharose CL-6B gel filtration chromatography and Mono Q HR 5/5 anion exchange chromatography. F-I had fairly good pH and thermal stability, within the range of pH 4.0 - 11.0 and below 55 ℃ , it demeaned fine β-glucosidase activity. The optimal pH and temperature of F-I were pH 5.0 and 55 ℃ respectively. EDTA, Zn^2＋ , and Cu^2＋ inhibited strongly against its activity. Specificity analysis of substratum indicated that F-I could highly hydrolyze artificial synthetically substratum of pNPG, it could also hydrolyze disaccharides such as cellobiose and gentiobiose that linked by β-glucoside bond, thus explained that it was a β-glucosidase. F-I acquitted itself well as fairly strong hydrolysis activity on GS, but had low hydrolysis activity on Rb2 and Rc. The pathway of hydrolysis of GS was Rb1→Rd→F2→C-K. The high effective hydrolysis of F-I on GS laid a foundation for rare GS Rb1 preparation industry.