芍药苷转化酶G6046的异源表达及酶活鉴定

Heterologous Expression of Paeoniflorin Converting Enzyme G6046 and the Identification of Its Enzymatic Activities

芍药苷是一种在传统草药芍药中发现的糖苷类化合物,对心脑血管系统有保护作用。然而,口服芍药苷的生物利用度较低,直接吸收的很少,极大地限制了其临床应用。已有研究表明,芍药苷的药理作用可能是由其代谢产物产生的,且主要在肠内菌中被代谢。本文研究了芍药苷转化酶G6046在大肠杆菌BL21 DE3中的诱导表达和纯化,并通过优化诱导条件提高酶的产量,发现在诱导表达的最佳条件(16℃,诱导24 h)下,菌体的产酶量可达到5.34 mg/g,产酶量提高51.69%。基于此诱导条件,本研究对G6046与芍药苷的反应条件(pH和温度)进行优化,发现G6046在pH 9.0、45℃时反应最快,酶活性最高,比酶活为14.56 U/mg。在此优化条件下扩大反应体系,将反应规模从G6046-底物(1/1,mg/mg)扩大到G6046-底物(1/20,mg/mg),转化产物的产量显著提升,为分离芍药苷转化产物奠定了基础。

Paeoniflorin is a glycoside compound found in Paeonia lactiflora Pall that shows various protective effects on the cardiocerebral vascular system and always used in traditional herbal medicine.However,the bioavailability of paeoniflorin by oral administration is low,which greatly limits its clinical application.It has been reported that the pharmacological effects of paeoniflorin might be generated by its metabolites and mainly metabolized in intestinal bacteria.In this paper,the induction expression and purification of paeoniflorin converting enzyme G6046 in Escherichia coli BL21 DE3 were studied.The induction reaction conditions were optimized to improve the enzyme yield and the optimal conditions for induction reaction were found to be 24 h induction reaction at 16℃.Enzyme production of bacterium reached 5.34 mg/g,representing a 51.69%increase in enzyme production.Based on the conditions of induction expression,the conditions(pH and temperature)for the reaction of G6046 with paeoniflorin were optimized,pH 9.0 and 45℃were proved to be the optimized conditions,resulting in a specific enzyme activity of 14.56 U/mg.The reaction system was enlarged under this optimized condition,and the reaction scale was from G6046-substrate(1∶1;mg/mg)to G6046-substrate(1∶20;mg/mg),the yield of the converted product significantly increased,which lays a foundation for the isolation of peonidin conversion product.