Azocasein Protease Activity Assay

Fifty μL of a 1% (w/v) azocasein solution were added to 50 μL of cell-free supernatant (containing protease) and the mixture was immediately incubated at 37 °C for 30 min. The reaction was stopped by mixing with 300 μL of 5% (w/v) trichloroacetic acid (TCA, Katayama Chemical, Osaka, Japan). Finally, the sample was centrifuged at 13,000 rpm for 10 min and 150 µL of the supernatant was mixed with an equal amount of 0.5 N NaOH. The absorbance of the mixture was measured at 450 nm using a Microplate Absorbance Reader (BIO-RAD, Hercules, CA, USA). One unit was defined as an increase of A_{450 nm} of 0.01 after incubation for 1 min [52 (link),53 (link),54 (link)].