A New Microplate Screening Method for the Simultaneous Activity Quantification of Feruloyl Esterases, Tannases, and Chlorogenate Esterases
A New Microplate Screening Method for the Simultaneous Activity Quantification of Feruloyl Esterases, Tannases, and Chlorogenate Esterases
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DOI：
10.1007/s12010-008-8319-8
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发表时间：
2008-12-01
影响因子：
3
通讯作者：
Mateos-Diaz, J. C.
中科院分区：
文献类型：
--
作者：
Ramirez, L.;Arrizon, J.;Mateos-Diaz, J. C.
阿魏酰、绿原酸酯酶和单宁酶是在药物相关的酚类修饰中有用的酶，作为几种退行性人类疾病的保护剂。因此，人们对发现这些酶的新来源越来越感兴趣。然而，传统的活性测量方法既耗时又不适合高通量筛选。在本研究中，一种新的微孔板高通量筛选方法成功地同时定量了上述所有活性。该方法可检测活性低至1.7 μ ml（-1）。此外，随着酶的添加量的增加，反应速率成比例地增加，并且没有发现对其他商业水解酶的干扰。同时对20株真菌生产动力学过程中获得的固体发酵提取物的阿魏酰、绿原酸酯酶和单宁酶活性进行了筛选，证明了该方法的实用性。其中7株至少有一种酯酶活性检测呈阳性。这一结果显示了快速常规筛选测定多种样品中，低至高的酶水平的潜力。
Feruloyl, chlorogenate esterases, and tannases are enzymes useful in phenolic modifications of pharmaceutical relevance as protectors against several degenerative human diseases. Therefore, there is a growing interest in discovering new sources of these enzymes. However, traditional methods for their activity measurements are time-consuming and poorly adapted for high-throughput screening. In this study, a successful new microplate high-throughput screening method for the simultaneous quantification of all mentioned activities is demonstrated. This method allows the detection of activities as low as 1.7 mU ml(-1). Furthermore, reaction rates increased proportionally with the amount of enzyme added, and no interferences with the other commercial hydrolases tested were found. The utility of the method was demonstrated after simultaneously screening feruloyl, chlorogenate esterase, and tannase activities in solid state fermentation extracts obtained during the kinetics of production of 20 fungal strains. Among these, seven strains were positive for at least one of the esterase activities tested. This result shows the potential for the rapid routine screening assays for multiple samples of moderate low to high enzymatic levels.