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

在本研究中，其目的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口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.以酶复合物的计算机模型为基础，以底物结合口袋中的4个氨基酸为靶标，通过SIMPLEX方法构建组合文库。利用模糊神经网络对初筛数据进行分析，得到了一些模糊规则。最后，证明了所得到的规则对于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-苯基丁酸酯仅显示出低的光学选择性。然后，在计算机上构建底物与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方法将限于疏水氨基酸（7种）的组合突变引入到所选择的位点。通过对SIMPLEX文库的筛选，得到了一些对映体选择性比野生型高的克隆。3.为了提高SIMPLEX方法的效率，提出了一种在乳胶微珠上扩增单DNA分子的新技术。在384孔板上进行一珠PCR后，可以在板上构建DNA文库。按照上述方法构建了该脂肪酶的突变库，并针对上述底物进行了筛选，从而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.