人参皂苷Rb3糖基水解酶的纯化及其反应特性

人参皂苷Rb3是三七茎叶皂苷的主要成分。为了充分利用廉价的三七茎叶皂苷,该研究以微生物Aspergillus sp. P90r菌为对象,综合运用生物转化的方法,经过提取、分离纯化和酶活力测定等步骤,最终以确定酶反应途径的方式得到了所产的特异性人参皂苷Rb3糖基水解酶的相关性质和动力学等反应特性。结果表明:该酶比Absidia sp. GRB3-X8r菌产酶活力高15%²5%,SDS-PAGE电泳结果测得分子量约为65.6ku,纯化后酶蛋白的含量为0.237 mg·mL^-1,蛋白比活力可达到169 U·mg^-1,纯化倍数为13.70,回收率为9.39%。人参皂苷Rb3糖基水解酶在pH=5.0的偏酸性环境下酶活力很高,最适反应条件:pH=3.0⁵.0,温度45℃,其中在pH=4.0⁶.0范围内相对稳定。该酶在20 min时进入混合级反应,酶反应米氏常数Km值为8.77 mmol·L^-1,V_max为57.44 mmol·L^-1

Purification and reaction characteristics of hydrolyzed ginsenoside Rb3-glycosylase

Hydrolyzed ginsenoside Rb3 is the main component of Panax notoginseng stem and leaf saponins.In order to make full use of cheap P.notoginsen g stem and leaf saponins,the microorganism Aspergillus sp.P90r was studied,and we used the biotransformation methods comprehensively.Related properties,kinetics and other reaction characteristics of the specific ginsenoside Rb3-glycosylase were produced by determining the way of enzymatic reaction through extraction,separation,purification,enzyme activity determination and other steps.The results were as follows:This enzyme activity was 15%-25%higher than the enzyme by Absidia sp.GRB3-X8r.The result of the molecular weight of enzyme protein was 65.6 ku by SDS-PAGE electrophoresis.The content of enzyme protein was 0.237 mg·mL^-1 after purification,the specific activity of the protein was attainable as 169 U·mg^-1,the purification factor was 13.70 and the recovery rate was 9.39%.And the enzyme activity of ginsenoside Rb3-glycosylase was higher in the pH 5.0 of acidic environment.The enzyme was suitable condition in the range of pH 3.0-5.0,in the temperature of 45℃,and it was relative stable in the range of pH 4.0-6.0.The enzyme entered the mixed-order reaction at 20 min,and the results of the enzyme reaction kinetics showed that the K m value was 8.77 mmol·L-1 and V max was 57.44 mmol·L-1·h-1.The reaction rate reached the maximum at 60 min,and the speed tended to be stable,and V max was 66.63 mmol·L-1·h-1.The results on catalytic properties of enzymes showed that the enzyme firstly hydrolyzes 20-O-xylose of ginsenoside Rb3,secondly hydrolyzes 3-O-glucosyl,eventually which the catalytic reaction products include the formed substance of F2 and C-K.In summary,the microbial Aspergillus sp.P90r enzyme has the specificity to hydrolyzed ginsenoside Rb3-xylose and Rb3-glucose.