气相色谱法测定脂肪酶的活力

Determination of lipase activity by gas chromatography

本文发展了一种以气相色谱检测脂肪酶活力的方法。以三丁酸甘油酯为底物,通过气相色谱对产物正丁酸进行定性定量分析。正丁酸在0.11～11.35mmol·L-1内线性关系良好,回归方程为:y=45464x-3115.2,r=0.9968。反应时间仅需5min,最适pH值和温度分别为7.5和32℃。低、中、高3个浓度正丁酸的回收率分别为90.3%,104.6%和89.4%;RSD分别为3.01%,4.50%和6.64%。通过实验测定出Km=0.25mmol·mL-1,奥利司他的半数抑制浓度(IC50)为0.0485mg·mL-1。本方法具有反应体积小、操作简单、快速、灵敏等特点。

A rapid gas chromatography method was developed for determination of lipase activity using tributyrin as substrate. The standard curves of butyric acid hydrolyzed from tributyrin were linear in the range of 0.11-11.35 mmol·L^-1. The recoveries of low, moderate and high concentrations of tributyrin were 90.3%, 104.6%, 89.4% with RSD of 3.01%, 4.50%, 6.64%, respectively. The incubation time was only 5 minutes which was less than with the half time of the conventional titrimetry and spectrophotometry. The optimum pH value was 7.5 and the optimum temperature was 32 ℃. Based on the Linweaver-Burk plots, the Michaelis- Menten constant was 0.25 mmol·mL^-1. The effect of orlistat on the enzyme inhibiting activity was studied to prove the accuracy of this method. It was found that the half-inhibition concentration （IC50） of orlistat was 0.048 5 mg·mL^-1. The small total reaction volume, the simple treating procedures, the high accuracy and precision present the advantages of the new method .