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

在这项研究中，它的目的是通过将组合突变引入到工业用脂肪酶的底物结合口袋中作为结构框架来创建具有多种底物特异性的脂肪酶群。结果概述如下1.根据酶复合体的计算机模型，用单纯形法建立了以底物结合袋中的4个氨基酸为靶标的组合库。利用模糊神经网络(FNN)对初筛获得的数据进行分析，并从中得出一些模糊规则。最后，所获得的规则被证明对获得新的突变酶是有用的。洋葱伯克霍尔德氏菌KWI-56脂肪酶对2-苯基丁酸酯的光学选择性很低。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，为引入突变提供可能的残基。在口袋中选择4个残基，并用单纯形法在选定的位点引入仅限于疏水氨基酸(7种)的组合突变。通过对SIMPLE文库的筛选，发现部分克隆具有比野生型更高的对映体选择性。3.为了提高SIMPLE方法的效率，建立了一种新的乳剂体系中微球上单个DNA分子的扩增技术。在384孔板上进行一次微珠聚合酶链式反应，即可构建DNA文库。构建了该脂肪酶的突变文库，并对上述底物进行了筛选，获得了一株对映选择性较高的突变体。

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.