Enhancement of the substrate specificity of D-amino acid oxidase based on tunnel-pocket engineering

D -Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of D -amino acids, making it one of the most promising routes for synthesizing optically pure L -amino acids, including L -phosphinothricin ( L -PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate D -PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis ( Rtw DAAO), and improved its catalytic potential toward D -PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M 3rd -SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M 3rd -SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M 3rd -SHVG reshapes the tunnel-pocket and corrects the direction of enzyme–substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M 3rd -SHVG as biocatalyst, 500 mM D, L -PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.