Zhongguo youzhi (May 2023)
基于酶反应动力学理论优化脂肪酶活力测定体系Optimization of lipase activity assay system based on enzyme reaction kinetics theory
Abstract
为了提高棕榈酸对硝基苯酯（p-NPP）法测定脂肪酶活力的灵敏度和准确度，以来源于Rhizopus oryzae的脂肪酶Lipase F-AP 15为研究对象，首先对其最适反应pH和反应温度进行优化，然后将表面活性剂作为激活因子引入酶活力测定体系，探究影响机制，并依据酶反应动力学理论优化测定条件(底物浓度、酶质量浓度、反应时间），对优化的p-NPP法的重复性和准确性进行了检验，最后以另一个来源的脂肪酶Lipase-PPL为模型，以同样思路与方法改进与优化其酶活力测定体系，以考察优化方法和思路的适用性。结果表明：表面活性剂PEG 8000的引入使得覆盖在脂肪酶活性位点的α-螺旋结构被重排而打开，脂肪酶由封闭构象转为并保持在活化构象，脂肪酶活性增强；优化测定体系的最适条件为pH 8.0、反应温度30 ℃、酶质量浓度1.00 mg/mL、底物浓度6 mmol/L、反应时间5 min，在此条件下测得的酶活力是常用p-NPP法（pH 8.0、反应温度30 ℃、酶质量浓度2.50 mg/mL、底物浓度10 mmol/L、反应时间8 min）测得酶活力的1.4倍；重复性和准确性试验显示，优化p-NPP法重复测定结果的相对标准偏差为1.21%,加标回收率为95.60%，均优于常用p-NPP法；优化方法和思路适用于其他来源的脂肪酶活力测定体系的优化。综上，通过将表面活性剂引入p-NPP法的脂肪酶活力测定体系，以酶反应动力学理论为指导优化测定条件，明显提高了方法的灵敏度，并有效保证了测定结果的重复性和准确性，缩短了检测时间，节省了检测成本。 In order to improve the sensitivity and accuracy of p-nitrophenol palmitate (p-NPP) method for the assay of lipase activity, the Lipase F-AP 15 from Rhizopus oryzae was taken as the research object, and the optimum reaction pH and reaction temperature were firstly optimized, then surfactant was introduced into the enzyme activity assay system as an activating factor, and the influence mechanism was explored. The assay conditions (substrate concentration, enzyme mass concentration, reaction time) were optimized according to the theory of enzyme reaction kinetics. The repeatability and accuracy of the optimized p-NPP method were examined. Finally, Lipase-PPL, another source of lipase, was used as a model to improve and optimize its enzyme activity assay system with the same idea and method to examine the applicability of the optimization method and idea. The results showed that with the introduction of surfactant PEG 8000, the α-helix structure covering the active site of lipase was rearranged and opened, and the lipase changed from closed conformation to and remained in the active conformation. The optimum conditions of the assay system were as follows: pH 8.0, reaction temperature 30 ℃, enzyme mass concentration 1.00 mg/mL, substrate concentration 6 mmol/L, and reaction time 5 min. Under the optimized conditions, the enzyme activity measured was 1.4 times that of the common p-NPP method (pH 8.0, reaction temperature 30 ℃, enzyme mass concentration 2.50 mg/mL, substrate concentration 10 mmol/L, and reaction time 8 min). The repeatability and accuracy experiments of the optimized p-NPP method showed that the relative standard deviation of the repeated assay results was 1.21%, and the recovery rate of standard addition was 95.60%, which was better than that of the common p-NPP method. The optimization method and idea was applicable to the optimization of lipase activity assay system from other sources. In conclusion, the introduction of surfactant in the lipase activity assay system of p-NPP method and the optimization of the assay conditions under the guidance of enzyme reaction kinetics theory can significantly improve the sensitivity of the method, effectively ensure the repeatability and accuracy of the assay results, shorten the assay time, and save the assay cost.
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