Phenylpyruvic acid (PPA) is a versatile organic acid extensively used in pharmaceuticals, food additives, and chemical synthesis. It serves as a key raw material for producing D-phenylalanine and phenyllactic acid, both important pharmaceutical intermediates. While chemical synthesis routes exist, they generate numerous by-products, create substantial environmental impact, and require demanding reaction conditions. The research focused on the L-amino acid deaminase from Proteus mirabilis, recognized for its broad substrate specificity and high catalytic efficiency. However, like many industrial enzymes, it suffers from substrate inhibition at concentrations above optimal levels.
Using molecular docking and computational modeling, researchers identified key residues surrounding the substrate-binding pocket that could influence substrate tolerance. Targeted residues were subjected to site-directed random mutagenesis, creating libraries of variants for screening. The screening identified a superior mutant PmiLAAD^M440V(M4). Structural and molecular dynamics analyses revealed that this mutation reshapes the substrate-binding pocket, altering substrate-enzyme interactions and alleviating the inhibitory effects of high substrate concentrations. Compared to the wild-type, the M4 variant increased product concentrations by 2.17-fold and 16.69-fold at substrate concentrations of 45 g/L and 60 g/L, respectively, demonstrating significantly enhanced catalytic efficiency and high-substrate tolerance.
The study provides both a practical biocatalyst for industrial PPA production and a strategic framework for addressing substrate inhibition in other enzyme systems. The researchers emphasize that binding pocket remodeling represents a powerful approach for optimizing industrial biocatalysts, with applications extending beyond amino acid deaminases to numerous enzymes facing similar limitations.
The work entitled “Enhanced substrate tolerance of L-amino acid deaminase by reshaping substrate binding pocket for phenylpyruvic acid production” was published on Systems Microbiology and Biomanufacturing (published on Mar. 17, 2026).

DOI: 10.1007/s43393-026-00448-6