α-L-鼠李糖苷酶RhaB2的异源表达和转化芦丁研究

Heterologous expression and rutin conversion study of α-L-rhamnose glycosidase RhaB2

中文关键词:  α-L-鼠李糖苷酶  芦丁  异槲皮素  异源表达  蛋白浓度

英文关键词:α-L-rhamnose glycosidase  rutin  isoquercitrin  heterologous expression  protein concentration

中文摘要:

      目的 探究α-L-鼠李糖苷酶RhaB2诱导表达水平及转化芦丁的条件, 提高对芦丁转化异槲皮素的转化率。方法 本研究通过单因素实验探讨了诱导温度、诱导时间和诱导剂浓度对酶表达量的影响, 并研究pH、温度、时间和加酶量4个方面对RhaB2转化芦丁条件的影响。结果 重组α-L-鼠李糖苷酶RhaB2在大肠杆菌BL21(DE3)中的最适表达条件为: 诱导温度28 ℃; 诱导时间20 h; IPTG浓度0.1 mmol/L。转化芦丁最适温度为50 ℃, 最适pH为5.0, 在此条件下反应2 h, 加酶量与底物的质量比为2:1时转化率为99.96%。结论 本研究成功构建了重组α-L-鼠李糖苷酶RhaB2的高效异源表达体系。在酶与底物质量比为2:1、反应2 h的工艺条件下, 芦丁转化率可达99.96%, 实现了异槲皮素的高效定向制备。

英文摘要:

      Objective To investigate the conditions for inducing expression levels of α-L-rhamnose glycosidase RhaB2 and its conversion of rutin, thereby enhancing the conversion rate of rutin to isoquercitrin. Methods Single-factor experiments examined the effects of induction temperature, duration and inducer concentration on enzyme expression levels. The effects of pH, temperature, incubation time and enzyme loading on RhaB2-mediated rutin conversion were also investigated. Results Optimal expression conditions for recombinant α-L-rhamnose glycosidase RhaB2 in Escherichia coli BL21(DE3) were: Induction temperature 28 °C; induction time 20 hours; IPTG concentration 0.1 mmol/L. The optimal temperature for rutin conversion was 50 °C, with an optimal pH of 5.0. Under these conditions, a 2-hour reaction at a 2:1 enzyme-to-substrate mass ratio achieved a conversion rate of 99.96%. Conclusion In this study, a highly efficient heterologous expression system for recombinant α-L-rhamnosidase RhaB2 is successfully constructed. Under the technological conditions where the mass ratio of the enzyme to the substrate is 2:1 and the reaction time is 2 hours, the conversion rate of rutin can reach 99.96%, thus enabling the highly efficient and targeted preparation of isorhamnetin.