固定化β-葡萄糖苷酶和蜗牛酶生物转化淫羊藿苷效率的比较研究

Transformation of icariin by immobilized β-glucosidase and snailase

本文制备了固定化β-葡萄糖苷酶和固定化蜗牛酶,优化二者转化淫羊藿苷的工艺条件并进行比较。利用交联-包埋法分别固定β-葡萄糖苷酶和蜗牛酶,单因素优化固定化酶的制备工艺,考察固定化酶的酶学性质。将固定化酶用于转化淫羊藿苷,考察转化温度、p H、酶与底物质量比、底物浓度、反应时间、使用次数对转化率的影响。游离酶与固定化酶显示出不同的酶学性质,固定化酶的热稳定性优于游离酶,4℃下贮藏4周,酶活保留约60%。固定化β-葡萄糖苷酶和固定化蜗牛酶转化淫羊藿苷的最优条件为:转化温度50℃,p H为5的磷酸缓冲液,酶与底物比为1∶1,底物质量浓度0.1 mg·m L^(-1),固定化β-葡萄糖苷酶转化6 h,固定化蜗牛酶转化2 h;两种固定化酶转化淫羊藿苷5次,平均转化率分别为70.76%和74.97%。β-葡萄糖苷酶和蜗牛酶固定化后,稳定性增强、使用寿命延长、可重复使用、转化淫羊藿苷的工艺简单和转化效率高,且固定化蜗牛酶转化淫羊藿苷的速率较快,适于转化淫羊藿苷的工业生产。

This study was performed to prepare immobilized β-glucosidase and snailase, then optimize and compare the process conditions for conversion of icariin. Immobilized β-glucosidase and snailase were prepared using crosslink-embedding method. The best conditions of the preparation process were optimized by single factor analysis and the properties of immobilized β-glucosidase and snailase were investigated. The reaction conditions including temperature, pH, substrate ratio, substrate concentration, reaction time and reusing times of the conversion of icariin using immobilized β-glucosidase or snailase were optimized. Immobilized β-glucosidase and snailase exhibited better heat stabilities and could remain about 60% activity after storage at 4℃ for 4 weeks. The optimized conditions for the conversion of icariin were as follows, the temperature of 50℃, pH of 5.0, enzyme and substrate ratio of 1 ： 1, substrate concentration of 0.1 mg·mL-1, reaction time of 6 h for β-glucosidase and 2 h for snailase, respectively. In 5 experiments, the average conversion ratio of immobilized β-glucosidase and snailase was 70.76% and 74.97%. The results suggest an effect of promoted stabilities, prolonged lifetimes in both β-glucosidase and snailase after immobilization. The immobilized β-glucosidase and snailase exhibited a higher conversion rate and reusability compared to the free β-glucosidase and snailase. Moreover, the conversion rate of immobilized snailase was higher than that of immobilized β-glucosidase. The process of icariin conversion using immobilized β-glucosidase and snailase was moderate and feasible, which suggests that immobilized enzymes may hold a promise for industrial usage.