Determination of Lipolytic Enzyme Activities
Determination of Lipolytic Enzyme Activities
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DOI：
10.1007/978-1-4939-0473-0_12
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发表时间：
2014-01-01
期刊：
影响因子：
--
通讯作者：
Kovacic, Filip
中科院分区：
文献类型：
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作者：
Jaeger, Karl-Erich;Kovacic, Filip
铜绿假单胞菌是一种多功能的人类机会致病菌，其产生并分泌大量的酶、蛋白质和小分子，其中许多作为毒力因子。值得注意的是，约40%的铜绿假单胞菌基因编码未知功能的蛋白质，其中超过80种编码推定的但仍然未知的脂解酶。这组水解酶（EC 3.1.1）已经知道了几十年，但只是最近，这些酶中的几个引起了人们的注意，作为潜在的毒力因子。可靠和可重复的酶活性测定对于确定其生理功能，特别是评估其对致病性的贡献至关重要。由于脂质的独特生物化学性质导致在水中形成胶束结构，底物乳液的可再现制备强烈依赖于所使用的方法。此外，相应底物乳液的物理化学性质可能会显著影响测试的脂解酶的活性。在这里，我们描述了脂肪酶，酯酶，磷脂酶和溶血磷脂酶的活性测定的常用方法。这些方法包括在体外用细胞提取物或分离的亚细胞区室以及用纯化的酶进行的脂解活性测定。我们试图描述标准化的协议，允许测定和比较来自不同来源的脂解酶的酶活性。这些方法还应鼓励假单胞菌群体解决由铜绿假单胞菌编码和产生的大量尚未开发的脂解酶。
Pseudomonas aeruginosa is a versatile human opportunistic pathogen that produces and secretes an arsenal of enzymes, proteins and small molecules many of which serve as virulence factors. Notably, about 40 % of P. aeruginosa genes code for proteins of unknown function, among them more than 80 encoding putative, but still unknown lipolytic enzymes. This group of hydrolases (EC 3.1.1) is known already for decades, but only recently, several of these enzymes have attracted attention as potential virulence factors. Reliable and reproducible enzymatic activity assays are crucial to determine their physiological function and particularly assess their contribution to pathogenicity. As a consequence of the unique biochemical properties of lipids resulting in the formation of micellar structures in water, the reproducible preparation of substrate emulsions is strongly dependent on the method used. Furthermore, the physicochemical properties of the respective substrate emulsion may drastically affect the activities of the tested lipolytic enzymes. Here, we describe common methods for the activity determination of lipase, esterase, phospholipase, and lysophospholipase. These methods cover lipolytic activity assays carried out in vitro, with cell extracts or separated subcellular compartments and with purified enzymes. We have attempted to describe standardized protocols, allowing the determination and comparison of enzymatic activities of lipolytic enzymes from different sources. These methods should also encourage the Pseudomonas community to address the wealth of still unexplored lipolytic enzymes encoded and produced by P. aeruginosa.