Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity
Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity
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高通量检测脂肪酶酯交换活性的荧光微孔板测定方法
DOI：
10.1016/j.ab.2018.03.010
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发表时间：
2018
影响因子：
2.9
通讯作者：
Wang Zhao
中科院分区：
文献类型：
--
作者：
Zheng Jianyong;Wei Wei;Lan Xing;Zhang Yinjun;Wang Zhao
本研究介绍了一种灵敏的荧光微板法检测脂肪酶的酯交换活性。以脂肪酶催化丁酰基4-甲基伞形酮(Bu-4-Mu)与甲醇间丁醇的酯交换反应为模型反应。通过检测λex330 nm和λem390 nm处的荧光强度来确定反应中4-甲基伞形酮(4-Mu)的释放。以几种脂肪酶为例考察了该方法的准确性和效率。计算了脂肪酶催化Bu-4-Mu与甲醇酯交换反应的表观米氏常数(Km)。该方法的主要优点是灵敏度高、试剂便宜、检测过程简单。
This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol intert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity atλex330 nm andλem390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process.