Engineering an α-Amino-ε-Caprolactam Racemase for Efficient d -Phenylalanine Synthesis Using a Coupled Colorimetric Screening Method

α-Amino-ε-caprolactam racemase (AoACLR) is a key biocatalyst for the synthesis of optically pure d-amino acids, yet its application is constrained by poor activity toward bulky aromatic substrates and limited thermostability. Here, we developed a coupled colorimetric high-throughput screening method and applied directed evolution, structure-guided engineering, and in silico screening to engineer AoACLR. Through combinatorial mutagenesis, we obtained M7 (N17D/L21V/L80T/S211G), which exhibited improved activity, soluble expression, and thermostability. Using l-phenylalaninamide as the substrate, the purified M7 exhibited approximately 1.89-fold higher specific activity toward l-phenylalaninamide than the previously reported L19V/L78T variant, together with 1.63-fold higher residual activity. In a coupled cascade biocatalytic system, M7 produced 14.61 g·L-1d-phenylalanine with 97.78% enantiomeric excess, corresponding to a 2.46-fold increase in d-phenylalanine production. Molecular docking and dynamics simulations suggested that M7 remodeled the substrate access pathway and strengthened the active-site interaction network. This work provides an improved AoACLR variant for efficient d-phenylalanine synthesis.