Development of a highly functional thermostable D-amino acid oxidase for efficient L-phosphinothricin biosynthesis...

D-amino acid oxidase (DAAO) has long been recognized as a promising catalyst for chiral amino acid biosynthesis owing to its pronounced stereospecificity. However, its limited thermostability restricts industrial utility and poses challenges for protein engineering. Herein, we engineered a DAAO variant, M5 (S42D/T84D/H138N/A193R/E195Y/C234L), via a ProteinMPNN-guided multiple codon saturation mutagenesis approach. This variant exhibited superior catalytic efficiency (27.3 mM^-1 s^-1, 29-fold relative to the starting enzyme) toward the non-natural compound D-glufosinate (D-PPT) and enhanced thermostability (519 min half-life at 50 °C, 208-fold relative to the starting enzyme). The evolution also expanded the enzyme's substrate scope toward several D-amino acids with different properties. The variant was proven able to completely transform 500 mM D-PPT in 7 h at a 100 mL scale with excellent stereospecificity. Molecular dynamics (MD) simulations gave the insight that the enhanced performance stems from altered surface charges and intramolecular/intermolecular networks. This strategy thus holds potential for engineering other enzymes involved in the biosynthesis of valuable products while reducing the screening costs during directed evolution.