人参皂苷Rb3糖基水解酶的纯化及其反应特性

Purification and reaction characteristics of hydrolyzed ginsenoside Rb3-glycosylase

关键词:  人参皂苷Rb3, 人参皂苷Rb3糖基水解酶, 酶学性质, 酶分离纯化, 酶促反应动力学

Key words:  hydrolyzed ginsenoside Rb3, hydrolyzed ginsenoside Rb3-glycosylase, enzyme properties, isolation and purification, enzyme kinetics

摘要:

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%,其SDS-PAGE电泳结果测得分子量约为65.6 ku,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的偏酸性的环境下酶活力很高,最适反应条件:pH=3.0～5.0,温度45 ℃,其中在pH=4.0～6.0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K_m值为8.77 mmol·L^-1,V_max为57.44 mmol·L^-1·h^-1,在60 min时反应速度达到最大,V_max趋于稳定,为66.63 mmol·L^-1·h^-1。通过对酶的催化特性研究表明,该酶先水解Rb3的20-O-木糖基,其次水解3-O-葡萄糖基,最终催化反应产物中有F2和C-K生成。综上结果,微生物Aspergillus sp. P90r菌酶具有能水解人参皂苷Rb3木糖基和葡萄糖基的特异性。

Abstract:

Hydrolyzed ginsenoside Rb3 is the main component of Panax notoginseng stem and leaf saponins. In order to make full use of cheap P. notoginseng 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.