脂肪酶活力测定中终止反应方法的比较分析

Comparative Analysis of Termination Reaction Methods in Lipase Activity Assay

关键词:  脂肪酶  酶活测定  终止反应  抑制效果

Key words:  lipase  activity assay  termination reaction  inhibiting effect

摘要:

[目的]寻找一种准确有效的脂肪酶活力测定中的终止反应方法,以进行脂肪酶酶学性质研究。[方法]比较分析前人研究脂肪酶常用的终止方法(无水乙醇法、EDTA法、三氯乙酸法、碳酸钠法、沸水浴法)对酶活力的抑制效果和吸光值的稳定性。[结果]无水乙醇法对酶活的抑制率为97.56%,终止反应后5min内净吸光值减少5%;EDTA法对酶活抑制率为32.94%,终止反应后10min内净吸光值增加5%;三氯乙酸法对酶活的抑制率为99.46%,终止反应后10min内净吸光值增加了0.47%;碳酸钠法对酶活抑制率为76.44%,终止反应后10min内净吸光值增加了0.53%;沸水浴法对酶活抑制率为99.51%,终止反应后10min内净吸光值减少0.77%,但沸水浴会导致剩余底物的大量分解。[结论]三氯乙酸法是一种准确有效的终止脂肪酶活力测定反应的方法,可用于脂肪酶酶学性质的研究。

Abstract:

[Objective] Searching an accurate and effective termination reaction method in lipase activity assay for studying lipase enzymatic characters.[Methods] Comparative analyses were conducted in inhibiting effect and result stability of termination methods(absolute ethanol, EDTA, trichloroacetic acid, sodium carbonate and boiling water bath), which predecessors used generally in lipase activity assay.[Results] Inhibition ratio of termination by absolute ethanol was 97.56%, and net absorbance reduced by 5% within 5min after termination; Inhibition ratio of terminaion by EDTA was 32.94%, and net absorbance increased by 5% within 10min after termination; Inhibition ratio of termination by trichloroacetic acid was 99.46%, and coloration net absorbance increased by 0.47% within 10min after termination; Inhibition ratio of termination by sodium carbonate was 76.44%, and net absorbance increased by 0.53% within 10min after termination; Inhibition ratio of termination by boiling water was 99.51%, and net absorbance reduced by 0.77% within 10min after termination, but it would result in the decomposition of most remaining substrates.[Conclusion] The termination method of trichloroacetic acid is an accurate and effective one in lipase activity assay, and can be adopt in analyzing lipase enzymatic characters.