Development of efficient nitrilase biocatalysts through growth selection for acetonitrile wastewater detoxification

Acetonitrile wastewater poses serious threat to both the ecological environment and human health. Conventional chemical hydrolysis of acetonitrile typically requires harsh conditions-elevated temperatures and strong acids or bases - leading to the generation of substantial undesirable by-products and inorganic waste. In contrast, nitrilase can directly hydrolyze nitriles groups into carboxylic acids in a single step, thereby minimizing by-product formation. In this study, a growth selection-based high-throughput screening method was established for screening nitrilase variants cultured in a medium with acetonitrile as the sole nitrogen source. This approach yielded two highly active mutants, designated C224R and 5MT, exhibiting 2.0-fold and 5.0-fold higher specific activity, respectively, compared to the wild-type enzyme. To enhance stability and reusability, the engineered variants were immobilized onto mesoporous silica carriers functionalized with 3-aminopropyldiethoxysilane. The immobilized enzymes exhibited significantly improved performance in degrading high-concentration acetonitrile wastewater and retain over 80% of their initial activity after 8 reuse cycles, highlighting their potential for practical wastewater treatment applications.