4.4. Assay of Nitrilase Activity

Nitrilase activity was determined in a 1020 μL reaction mixture at 28 °C. For E. coli cells, 20 μL acrylonitrile was added into 1 mL resuspended cells with OD₆₀₀ = 5.0 in 0.1 M PBS buffer (pH 7.5). For R. ruber cells, 20 μL acrylonitrile was added into 1 mL of a mixture containing 200 μL resuspended cells with OD₄₆₀ = 40.0 in 0.1 M PBS and 800 μL of 0.1 M PBS buffer (pH 7.5) [26]. The reaction was terminated by adding 100 μL of 3 M HCl. The mixture was centrifuged at 13,000 rpm for one minute, and the supernatant was used to analyze the ammonium acrylate concentration via gas chromatography (Abel Industries Canada Ltd., Vancouver BC Canada, AB-INOWAX 30 m × 0.25 mm × 0.25 μm). Acetamide was used as the internal standard. The GC operation conditions were as follows: a TRACE 1300 GC device was equipped with a polyethylene glycol polymer capillary column (30 m × 0.25 mm × 0.25 μm) and a flame ionization detector—the injection and detector temperatures were 260 °C, while the column temperature was 190 °C.

One unit (U) of nitrilase activity was defined as the quantity of ammonium acrylate (μmol) that was catalyzed by 1 mg DCW (DCW, dry cell weight) cell suspension in one minute.