Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.
J Biotechnol. 2011 Oct 20;156(1):1-10. doi: 10.1016/j.jbiotec.2011.08.003. Epub 2011 Aug 10.
We performed a laboratory evolution study with the epoxide hydrolase from Aspergillus niger M200. This enzyme exhibits no enantioconvergence with the substrates styrene oxide or para-chlorostyrene oxide, i.e. racemic vicinal diols are produced from the racemic substrates. After saturation mutagenesis, screening by chiral gas chromatography revealed enzyme variants with improved enantioconvergence as manifested by an increased enantiomeric excess of the diol product. Nine amino acid exchanges accumulated in the active site and the substrate access tunnel over the course of 5 productive rounds of iterative saturation mutagenesis, resulting in an enantioconvergent epoxide hydrolase variant. The final mutant enzyme transformed racemic styrene oxide and para-chlorostyrene oxide to (R)-diol enantiomers with enantiomeric excesses of 70%. Sequential bi-enzymatic reactions using the wild-type EH and/or its evolved variants enabled preparation of the chiral building blocks (R)-phenyl-1,2-ethanediol and (R)-para-chlorophenyl-1,2-ethanediol from inexpensive racemic epoxides with enantiomeric excesses of 91% and 88%, respectively.
我们使用黑曲霉 M200 的环氧化物水解酶进行了实验室进化研究。该酶与底物苯乙烯氧化物或对氯苯乙烯氧化物没有对映体收敛性，即从外消旋底物中产生外消旋的偕二醇。经过饱和诱变，手性气相色谱筛选揭示了具有改善的对映体收敛性的酶变体，表现在二醇产物的对映体过量增加。在 5 轮迭代饱和诱变过程中，在活性位点和底物进入隧道中积累了 9 个氨基酸取代，导致对映体收敛的环氧化物水解酶变体。最终的突变酶将外消旋的苯乙烯氧化物和对氯苯乙烯氧化物转化为（R）-二醇对映体，对映体过量分别为 70%。使用野生型 EH 和/或其进化变体的连续双酶反应，能够从廉价的外消旋环氧化物制备手性砌块（R）-苯基-1,2-乙二醇和（R）-对氯苯基-1,2-乙二醇，对映体过量分别为 91%和 88%。
