发酵液中酮基还原酶活性测定方法的构建

A method for determining ketoreductase activity in broth

本研究以4-氯乙酰乙酸乙酯(COBE)为底物,还原型辅酶Ⅱ(NADPH)为辅酶,应用紫外-可见光分光光度计法测定发酵液中酮基还原酶活性,通过连续测定NADPH消耗量来计算酶活力,得到了最佳反应体系:NADPH浓度为0.2 mmol/L,COBE浓度为1.0 mmol/L,磷酸缓冲溶液浓度为100 mmol/L、p H值为6,反应温度为40℃。在此条件下平行检测5次酶活,相对标准偏差(RSD)为0.48%。此方法操作简便,耗时短,精确度高,重复性好,可作为发酵液中酮基还原酶活性测定方法加以推广。

The UV-Vis spectrophotometer method to determine the enzyme activity in fermentation broth was studied using ethyl 4-chloroacetoacetate （COBE） as substrate,nicotinamide adenine dinucleotide hydro-phosphate acid （NADPH） as a coenzyme.The enzyme activity was calculated by continuously measuring the consumption of NADPH.The optimum reaction conditions were acquired as follows：NADPH concentration 0.2 mmol/L,COBE 1.0 mmol/L,phosphate buffer 100 mmol/L,pH 6 and temperature 40 ℃.Finally,five times parallel detections were done to determine the enzyme activity under the optimum reaction system.It showed that relative standard deviation was 0.48％.This method with advantages of simple,short time-consuming,high accuracy,good repeatability could be generally used in the measurement of ketoreductase activity.