Engineered Nitrilase-Mediated Regioselective Hydrolysis of 4-Cyanobenzonitrile for Efficient Synthesis of 4-Cyanobenzoic Acid

As a pivotal synthetic intermediate for pharmaceuticals, agrochemicals, and advanced materials, 4-cyanobenzonitrile has garnered significant interest. However, conventional chemical approaches face inherent limitations in achieving regioselective hydrolysis of its symmetrical cyano groups to produce 4-cyanobenzoic acid, primarily due to harsh reaction conditions and poor selectivity. We predicted the kinetic parameters (k_cat value) of 30 nitrilases from different sources by machine learning model and five nitrilases were chosen for further researches. Subsequently, to address this challenge, we heterologously expressed and characterized two nitrilases—NIT-3 from Fusarium fujikuroi IMI 58289 and NIT-4 from Paraburkholderia graminis—for biocatalytic hydrolysis under industrially relevant high substrate loading. Remarkably, in a 100-L bioreactor system, both enzymes demonstrated exceptional catalytic efficiency, utilizing 7.50 g/L (DCW, dry cell weight) whole-cell biocatalysts to completely hydrolyze 120 g/L 4-cyanobenzonitrile within 8 h, achieving > 99% conversion and 100% regioselectivity toward 4-cyanobenzoic acid. The robust substrate tolerance, operational stability, and scalability of these nitrilases underscore their potential for sustainable industrial synthesis of high-value aromatic carboxylic acids.

Graphical Abstract