Remodeling of bacterial nitrilase active pocket improves the capability to degrade glucosinolate-derived nitriles.

Nitriles exhibit acute cytotoxicity to human and animal cells. Nitrilase is a green biocatalyst that can directly convert nitrile into nontoxic carboxylic acids and ammonia. However, the nitrilases capable of degrading 3-butenenitrile and 4-pentenenitrile derived from glucosinolate present in rapeseed meals are still limited. This study expressed the nitrilase 2 gene from bacterium Paraburkholderia graminis (PgNIT2) in Escherichia coli. The purified recombinant nitrilase rPgNIT2 showed specific activities of 5.94 U/mg and 6.66 U/mg when using 3-butenenitrile and 4-pentenenitrile as substrates at the optimal pH 7.0 and temperature 45 °C. The substrate binding pocket of PgNIT2 comprising 12 amino acids was required for rPgNIT2 activity. Remarkably, the specific activity of mutant enzyme A190I increased by 68.3% and 57.3%, respectively, against 3-butenenitrile and 4-pentenenitrile compared with the wild-type enzyme. Furthermore, the A190I showed improved thermostability and hydrolysis ability towards 3-butenenitrile and 4-pentenenitrile compared with rPgNIT2 but shared the same optimal pH and temperature. These results provided theoretical guidance for engineering of bacterial nitrilases used for rapeseed meal detoxification.