Azocasein Proteolytic Activity Assay

The proteolytic activity was assessed by mixing 1 mL of culture supernatant and 1 mL of 1% (w/v) azocasein solution dissolved in 0.2 M Tris–HCl buffer of pH 7.0. The enzyme–substrate reaction was allowed to proceed for 30 min at 37 °C and was ended through the addition of 2 mL of 10% (w/v) trichloroacetic acid solution. Thence, incubation for 60 min in a crushed ice bath. The amount of soluble degradation proteins (C) was measured (mg/mL) following this calculation; C (mg/mL) = 1.55 OD₂₈₀ − 0.76 OD₂₆₀. One unit (1 U) of proteolytic enzyme activity was equivalent to one microgram of released l-tyrosine per milliliter per minute of the reaction at the standard conditions of experimentations.