Department of Chemical and Biological Engineering, Institute of Bioengineering, Zhejiang University, Hangzhou 310027, People's Republic of China.
Appl Microbiol Biotechnol. 2013 Jun;97(11):4897-906. doi: 10.1007/s00253-012-4396-2. Epub 2012 Sep 19.
The present work created an esterase variant from Rhodobacter sphaeroides (RspE) with enhanced selectivity in hydrolytic kinetic resolutions by directed evolution. A "model" substrate, methyl mandelate, was introduced in the high-throughput screening procedure. E values of a variant CH (Asn62Cys/Leu145His) for six different esters were 10-83, which were a relative improvement compared to 2-20 for the wild type. Our subsequent crystal structure interpretation and molecular dynamics simulations helped shed light on the source of enantioselectivity modified by directed evolution. Though mutations displayed no "direct" interaction with the substrate, they were hypothesized to strengthen the intramolecular interaction in the catalytic cavity of variant. Conformation analysis revealed that the enhanced enantioselectivity of variant CH for the seven substrates applied in this study was derived from the decrease in size of the substrate binding pocket.
本工作通过定向进化从球形红杆菌（Rhodobacter sphaeroides）中创建了一种酯酶变体，该变体在水解动力学拆分中具有增强的选择性。“模型”底物扁桃酸甲酯被引入高通量筛选程序中。变体 CH（Asn62Cys/Leu145His）对六种不同酯的 E 值为 10-83，与野生型的 2-20 相比有相对提高。我们随后的晶体结构解释和分子动力学模拟有助于阐明定向进化修饰对立体选择性的来源。尽管突变与底物没有“直接”相互作用，但它们被假设可以增强变体催化腔中分子内相互作用。构象分析表明，变体 CH 对本研究中应用的七种底物的增强的对映选择性源自底物结合口袋的尺寸减小。
