Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576.
Chem Commun (Camb). 2012 May 14;48(38):4618-20. doi: 10.1039/c2cc30779k. Epub 2012 Mar 19.
Directed evolution of a monooxygenase to achieve very high enantioselectivity for hydroxylation at non-activated carbon atoms is demonstrated for the first time, where a triple mutant of P450pyr hydroxylase is obtained via determination of enzyme structure, iterative saturation mutagenesis, and high-throughput screening with a MS-based ee assay to increase the product ee from 53% to 98% for the hydroxylation of N-benzyl pyrrolidine to (S)-N-benzyl 3-hydroxypyrrolidine.
首次实现了单加氧酶的定向进化，使其在非活化碳原子的羟化反应中具有极高的对映选择性，方法是通过测定酶结构、反复饱和突变和基于 MS 的 ee 测定的高通量筛选，获得 P450pyr 羟化酶的三重突变体，从而将 N-苄基吡咯烷的羟化产物的对映体过量值从 53%提高到 98%。
