Department of Microbiology, Faculty of Biology, University of Barcelona, Av. Diagonal 645, Barcelona, Spain.
Biochimie. 2010 Mar;92(3):307-16. doi: 10.1016/j.biochi.2009.11.005. Epub 2009 Nov 26.
Growth of Pseudomonas sp. 42A2 on oleic acid releases polymerized hydroxy-fatty acids as a result of several enzymatic conversions that could involve one or more lipases. To test this hypothesis, the lipolytic system of strain Pseudomonas sp. 42A2 was analyzed, revealing the presence of at least an intracellular carboxylesterase and a secreted lipase. Consensus primers derived from a conserved region of bacterial lipase subfamilies I.1 and I.2 allowed isolation of two secreted lipase genes, lipA and lipC, highly homologous to those of Pseudomonas aeruginosa PAO1. Homologous cloning of the isolated lipA and lipC genes was performed in Pseudomonas sp. 42A2 for LipA and LipC over-expression. The overproduced lipases were further purified and characterized, both showing preference for medium fatty acid chain-length substrates. However, significant differences could be detected between LipA and LipC in terms of enzyme kinetics and behaviour pattern. Accordingly, LipA showed maximum activity at moderate temperatures, and displayed a typical Michaelis-Menten kinetics. On the contrary, LipC was more active at low temperatures and displayed partial interfacial activation, showing a shift in substrate specificity when assayed at different temperatures, and displaying increased activity in the presence of certain heavy metal ions. The versatile properties shown by LipC suggest that this lipase could be expressed in response to variable environmental conditions.
假单胞菌 42A2 在油酸上的生长会释放出聚合羟基脂肪酸，这是由于几种酶促转化的结果，其中可能涉及一种或多种脂肪酶。为了验证这一假设，分析了假单胞菌 42A2 的脂肪酶系统，结果表明至少存在一种细胞内羧酸酯酶和一种分泌脂肪酶。源自细菌脂肪酶亚家族 I.1 和 I.2 的保守区域的通用引物允许分离出两种分泌脂肪酶基因，lipA 和 lipC，它们与铜绿假单胞菌 PAO1 的高度同源。在假单胞菌 42A2 中进行了分离的 lipA 和 lipC 基因的同源克隆，以实现 LipA 和 LipC 的过表达。过表达的脂肪酶进一步进行了纯化和特性分析，两者均优先选择中等脂肪酸链长的底物。然而，在酶动力学和行为模式方面，可以检测到 LipA 和 LipC 之间的显著差异。因此，LipA 在中等温度下表现出最大活性，并显示出典型的米氏动力学。相反，LipC 在低温下更活跃，并表现出部分界面激活，当在不同温度下进行测定时，显示出底物特异性的变化，并在存在某些重金属离子时显示出增加的活性。LipC 表现出的多功能特性表明，这种脂肪酶可以响应环境条件的变化而表达。
