响应面法优化淀粉葡糖苷酶的酶反应体系

Optimization of Enzyme Reaction System for Amyloglucosidase by Response Surface Methodology

为建立并优化淀粉葡糖苷酶的酶反应体系,通过单因素分析和响应面实验设计,以淀粉葡糖苷酶能够直接作用的天然底物即淀粉为作用对象,以反应速度为指标,考察可溶性淀粉浓度、淀粉葡糖苷酶浓度、反应时间3个因素对酶促反应速度的影响。结果表明,所建立淀粉葡糖苷酶的最佳酶反应体系为可溶性淀粉质量浓度10.30 mg/mL、淀粉葡糖苷酶浓度1.15 U/mL、反应时间7.00 min,此条件下验证得酶促反应速度可达(15.17±1.84)mmol/(L·min),与理论预测值相对误差为1.69%。用响应面法建立的模型预测值与实际值的吻合度较高,说明淀粉葡糖苷酶酶反应体系具有实际意义,可应用于有效筛选糖苷酶抑制剂等相关的研究中,以便获得可信度更高的抑制数据。

In order to establish and optimize the enzyme reaction system of amyloglucosidase,through single-factor optimization and Box-Behnken experiment design,the author used a natural substrate-starch that amyloglucosidase could directly act on as the target,took reaction rate as an indicator to explore the influences of soluble starch concentration,amyloglucosidase concentration and reaction time on the reaction rate of amyloglucosidase on substance.The results showed that the enzyme reaction rate of amyloglucosidase reached(15.17±1.84)mmol/(L·min)under its optimal enzyme reaction system with 10.30 mg/mL soluble starch,1.15 U/mL amyloglucosidase,and 7.00 min reaction time,whose relative error with theoretical predictions in the obtained model is 1.69%.The predicted values of the enzymatic reaction rates of the above model were highly consistent with the actual results,indicating that the established reaction system could predict the actual enzyme activity commendably.Hence the system of amyloglucosidase by response surface methodology is reliable and can be used effectively in related researches such as screening out the glycosidase inhibitors effectively to obtain more reliable data.