Hydrophilicity‐Based Engineering of the Active Pocket of D‐Amino Acid Oxidase Leading to Highly Improved Specificity toward D‐Glufosinate

Abstract Due to its stringent stereospecificity, D‐amino acid oxidase (DAAO) has made it very easy to synthesize L‐amino acids. However, the low activity of the wild‐type enzyme toward unnatural substrates, such as D‐glufosinate (D‐PPT), restricts its application. In this study, DAAO from Rhodotorula gracilis ( Rg DAAO) was directly evolved using a hydrophilicity‐substitution saturation mutagenesis strategy, yielding a mutant with significantly increased catalytic activity against D‐PPT. The mutant displays distinct catalytic properties toward hydrophilic substrates as compared to numerous WT‐DAAOs. The analysis of homology modeling and molecular dynamic simulation suggest that the extended reaction pocket with greater hydrophilicity was the reason for the enhanced activity. The current study established an enzymatic synthetic route to L‐PPT, an excellent herbicide, with high efficiency, and the proposed strategy provides a new viewpoint on enzyme engineering for the biosynthesis of unnatural amino acids.