Microenvironment Synergistic Engineering Enables Efficient Nitrilase-Catalyzed Amino Acid Synthesis from Short-Chain Nitriles

Nitrilases are attractive biocatalysts for green chemical synthesis, yet their catalytic efficiency toward short-chain aliphatic nitriles remains insufficient for sustainable amino acid production. To address this limitation, we developed a microenvironment synergistic engineering (MSE) strategy and applied it to the newly identified nitrilase BbNit. This approach integrated active site structural analysis, flexible loop dynamics, and coevolutionary residue profiling to construct an 11-site focused mutant library. Screening identified the double mutant L194W/V199K, which displays a pronounced shift in substrate specificity, with its specific activity toward methylaminoacetonitrile increasing from 6.64 to 55.18 U·mg–1. The catalytic efficiency expressed as Kcat/Km increased by 5.6-fold, while activity toward aromatic nitriles was significantly reduced. Molecular dynamics simulations indicated that the synergistic mutations reinforced local structural rigidity and promoted active site preorganization, shortening the nucleophilic attack distance. Furthermore, a whole-cell biocatalyst based on the optimized mutant enabled high-titer amino acid biosynthesis, producing 78.51 g·L–1 sarcosine and 237.72 g·L–1 iminodiacetic acid with near complete conversion in fed-batch reactions. Collectively, this work establishes MSE as a semi rational design paradigm and provides a biocatalytic platform for efficient amino acid synthesis from short-chain aliphatic nitriles.