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

在这项研究中，它的目标是通过将组合突变仅引入工业上有用的脂肪酶的底物结合袋作为结构框架来创建具有多种底物特异性的脂肪酶组。结果概要如下。以酶配合物的计算机模型为基础，采用SIMPLEX方法构建了以底物结合袋中的4个氨基酸为靶标的组合文库。采用模糊神经网络（FNN）对首次筛选中获得的数据进行分析，并从中得到一些模糊规则。结果表明，所得规律可用于获得新的突变酶。洋葱伯克霍尔德菌KWI-56脂肪酶对2-苯基丁酸盐只有较低的光学选择性。然后，在计算机上构建底物与酶之间反应中间体的结构模型，给出引入突变的可能残基。在口袋中选择4个残基，通过SIMPLEX方法将7种疏水氨基酸引入所选位点进行组合突变。对SIMPLEX文库的筛选使部分克隆的对映体选择性高于野生型。为了使SIMPLEX方法更加有效，开发了一种在乳剂微珠上扩增单个DNA分子的新技术。384孔板上PCR 1个头后，可在板上构建DNA文库。如上所述构建了脂肪酶的突变文库，并针对上述底物进行筛选，获得了高对映选择性的突变体。

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.