Site-directed mutagenesis studies of the aromatic residues at the active site of a lipase from Malassezia globosa
Site-directed mutagenesis studies of the aromatic residues at the active site of a lipase from Malassezia globosa
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DOI：
10.1016/j.biochi.2014.02.004
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发表时间：
2014-07-01
期刊：
影响因子：
3.9
通讯作者：
Wang, Yonghua
中科院分区：
文献类型：
--
作者：
Gao, Chongliang;Lan, Dongming;Wang, Yonghua
球形马拉色菌脂肪酶（SMG1）对单酰甘油和二酰甘油具有特定活性，但对三酰甘油没有活性。对SMG1结构的结构分析表明，两个庞大的芳香族残基W116和W229位于活性位点的入口处。为了研究这两个残基在底物识别和催化反应中的功能，将它们突变为一系列氨基酸。随后，对这些突变体的生化特性进行了研究。尽管活性降低，但W229L和W116A在底物偏好性上有显著变化。W229L对短链底物的偏好性增加，而W116A对长链底物有偏好。此外，W116A和W116H在45℃时的半衰期分别为346.6分钟和115.5分钟，与天然酶相比有显著提高。而且，W116A、W116F和W229F突变体的最适底物从对硝基苯辛酸酯转变为对硝基苯肉豆蔻酸酯。这些发现不仅阐明了脂肪酶的结构/功能关系，还为潜在的工业应用奠定了基础。（C）2014爱思唯尔马松公司。版权所有。
The lipase from Malassezia globosa (SMG1) has specific activity on mono- and diacylglycerol but not on triacylglycerol. The structural analysis of SMG1 structure shows that two bulky aromatic residues, W116 and W229, lie at the entrance of the active site. To study the functions of these two residues in the substrate recognition and the catalytic reaction, they were mutated to a series of amino acids. Subsequently, biochemical properties of these mutants were investigated. Although the activities decrease, W229L and W116A show a significant shift in substrate preference. W229L has an increased preference for short-chain substrates whereas W116A has preference for long-chain substrates. Besides, the half-lives of W116A and W116H at 45 degrees C are 346.6 min and 115.5 min respectively, which improve significantly compared to that of native enzyme. Moreover, the optimum substrate of W116A, W116F and W229F mutants shifted from p-nitrophenyl caprylate to p-nitrophenyl myristate. These findings not only shed light onto the lipase structure/function relationship but also lay the framework for the potential industrial applications. (C) 2014 Elsevier Masson SAS. All rights reserved.