Sequential biosynthesis of 6‐ and 2‐chloronicotinic acids using wild‐type nitrilase and its engineered variant

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Chloronicotinic acids (CAs) are high‐value fine chemical intermediates for pharmaceuticals and agrochemicals. Chemical hydrolysis of mixed chloronicotinonitriles (CNs) derived from industrial raw materials suffers from poor substrate selectivity, complicated downstream processing, and low yield. To address these challenges, a sequential biosynthesis route was established for 6‐CA and 2‐CA. The nitrilase from Paraburkholderia graminis ( Pg NIT) exhibited strict selectivity and high activity toward 6‐CN, while a mutant Pg NIT‐9M with enhanced catalytic efficiency for 2‐CN was obtained via computer‐assisted design. The two enzymes were applied for stepwise bioproduction of 6‐CA and 2‐CA from a 2‐CN/6‐CN mixture. As a result, complete utilization of the mixed CN substrates was achieved, with the reaction yields of both 6‐CA and 2‐CA exceeding 99%. High‐purity 6‐CA and 2‐CA were obtained after further purification. This strategy realized green and efficient conversion of industrial mixed CNs, reduced resource waste and environmental pollution, and provided a sustainable biocatalytic route for CA production.