Improving the catalytic activity of lipase LipK107 from Proteus sp. by site-directed mutagenesis in the lid domain based on computer simulation
Improving the catalytic activity of lipase LipK107 from Proteus sp. by site-directed mutagenesis in the lid domain based on computer simulation
复制标题
提高变形杆菌脂肪酶 LipK107 的催化活性。
DOI：
10.1016/j.molcatb.2010.12.001
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发表时间：
2011-03-01
影响因子：
--
通讯作者：
Wei, Dongzhi
中科院分区：
文献类型：
--
作者：
Gao, Bei;Xu, Tao;Wei, Dongzhi
研究了变形杆菌脂肪酶LipK 107催化外消旋体动力学拆分的能力。以外消旋1-苯基乙醇在有机介质中的拆分为模型反应。该转化率是显着依赖于水含量和LipK 107显示在很宽的范围内的水含量没有明显的损失酶的对映体歧视的高活性。此外，酰基供体的链长对转化率也有显著影响，棕榈酸甲酯的对映选择性最高。基于LipK 107的计算机模型结构分析，在盖区引入不同的突变。表达、纯化LipK 107的每种衍生物，并评估其活性。根据预测结果，分别以突变体E130 L + K131 I和T138 V为催化剂，1-苯乙醇的转化率有较大提高，但对映体区分度略有增加。此外，还测定了盖的疏水性和静电性对脂肪酶活性的影响。这一结果表明，对盖的修饰可以显著提高酶的活性和催化反应的产率，并可应用于其他脂肪酶。计算机模拟将使识别氨基酸替换的过程更有效。(C)2010爱思唯尔有限公司版权所有。
The capacity of lipase LipK107 from Proteus sp. catalyzing the kinetic resolution of racemates was investigated. The resolution of racemic 1-phenylethanol in organic medium was selected as model reaction. The conversion was dramatically dependent on the water content and the LipK107 showed high activity in a wide range of water content without appreciable loss of enzyme enantiodiscrimination. Besides, the chain length of acyl donor also had a significant effect on the conversion, and the highest enantioselectivity was achieved when methyl palmitate was used. Based on the analysis of computer model structure of LipK107, different mutations were introduced into the lid region. Each derivative of LipK107 was expressed, purified, and assessed of the activity. According to the prediction, using mutants El 30L + K131I and T138V as catalyst, respectively, the conversions of 1-phenylethanol improved greatly with a slight increase of enantiodiscrimination. In addition, the effects of hydrophobicity and electrostatic of the lid on lipase activity were determined. This work indicated that the modification of the lid might considerably enhance the activity and improve the yield of catalytic reactions, which could apply to other lipases. The computer simulations would make the process of identifying amino acids for substitution efficiently. (C) 2010 Elsevier B.V. All rights reserved.