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Comprehensive analysis of the substrate-binding pocket relating to enantilselectivity of enzyme

与酶对映体选择性相关的底物结合口袋的综合分析

基本信息

批准号：
16360411
负责人：
NAKANO Hideo
金额：
$ 9.73万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

In this research, it has aimed to create the lipase group that has a variety of substrate specificities by introducing the combinatorial mutation only into the substrate binding pocket of an industrially useful lipase as a structural frame. The outline of the result is shown as follows.1. The combinatorial library where four amino acid in the substrate binding pocket had been made a target based on the computer model of the enzyme complex was made by the SIMPLEX method. Acquired data in the first screening was analyzed by fuzzy neural net work (FNN), and some fuzzy rules were derived from the analysis. Finally, the obtained rules were proven to be useful in obtaining new mutant enzymes.2. The Burkholderia cepacia KWI-56 lipase shows only a low optical selectivity to 2-phenylbutyrate. Then, the structural model of the reaction intermediate between the substrate and the enzyme was constructed on the computer to give possible residues for introducing mutations. Four residues were selected in the pocket, and combinatorial mutation limited to the hydrophobic amino acids (seven kinds) was introduced to the selected site by the SIMPLEX method. The screening of the SIMPLEX library gave some clones showing a higher enantioselectivity than the wild type.3.To make the SIMPLEX method more effective, a new technology amplifying single DNA molecules on microbeads in emulsion was developed. After one bead PCR on a 384-well plate, DNA library could be constructed on the plate. The mutation library of the lipase was constructed as above, and screened against the above-mentioned substrate, resulting that a high enantioselecitve mutant was obtained.

在这项研究中，其目的是通过仅将组合突变引入工业上有用的脂肪酶的底物结合口袋作为结构框架，创建具有多种底物特异性的脂肪酶基团。结果概要如下： 1．基于酶复合物的计算机模型，通过SIMPLEX方法制作了以底物结合袋中的四个氨基酸为靶标的组合文库。通过模糊神经网络（FNN）对第一次筛选中获得的数据进行分析，并从分析中得出一些模糊规则。最后证明所得到的规则对于获得新的突变酶是有用的。 2．洋葱伯克霍尔德菌 KWI-56 脂肪酶对 2-苯基丁酸仅表现出较低的光学选择性。然后，在计算机上构建底物和酶之间的反应中间体的结构模型，以给出引入突变的可能残基。在口袋中选择4个残基，并通过SIMPLEX方法将仅限于疏水性氨基酸(7种)的组合突变引入到所选位点。通过SIMPLEX文库的筛选，得到了一些比野生型具有更高对映选择性的克隆。3.为了使SIMPLEX方法更加有效，开发了一种在乳液中微珠上扩增单个DNA分子的新技术。在384孔板上进行一珠PCR后，即可在板上构建DNA文库。如上所述构建脂肪酶突变文库，并针对上述底物进行筛选，得到高对映选择性突变体。