Biotransformation and Biocatalysis, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Chembiochem. 2020 Jul 1;21(13):1893-1904. doi: 10.1002/cbic.201900726. Epub 2020 Mar 5.
The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis-stilbene oxide, and enantiocomplementary mutants produced (S,S)- and (R,R)-stilbene diol with >97 % enantiomeric excess. An (R,R)-selective mutant was used to prepare (R,R)-stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee). Some variants displayed higher catalytic rates (k ) than the original enzyme, but in most cases K values increased as well. The results demonstrate the feasibility of computational design and screening to engineer enantioselective epoxide hydrolase variants with very limited laboratory screening.
在制备性生物催化中使用酶时，通常需要通过蛋白质工程来调整酶的选择性。本文探索了使用计算文库设计和分子动力学模拟来创建具有从内消旋环氧化物生产对映二醇的选择性的柠檬烯环氧化物水解酶变体。针对三种不同大小的底物：顺式-2,3-丁烯氧化物、环戊烯氧化物和顺式-亚乙烯基氧化物。测试的 28 个设计中的大多数都是活性的，并表现出预测的对映选择性。对于体积庞大的底物顺式-亚乙烯基氧化物，获得了极好的对映选择性，并且产生了对映体互补的突变体，具有 >97%的对映过量（ee）。使用（R,R）选择性突变体以高对映体纯度（98%转化为二醇，>99%ee）制备（R,R）-亚乙烯基二醇。一些变体显示出比原始酶更高的催化速率（k），但在大多数情况下 K 值也增加了。结果表明，通过计算设计和筛选来工程具有非常有限的实验室筛选的对映选择性环氧化物水解酶变体是可行的。
