Protease activity was indicated by skim milk degradation in terms of clear zones of 1% skim milk- NA plates (1% skim milk solution mixed at 45 °C with NA) after 24 h of incubation at 30 °C.
Protease activity was assayed according to Dumusois and
Priest, 1993. Azocasein (0.2% w/v in 0.1 M phosphate buffer, pH 8)
1 ml, and enzyme source (0.5 ml) were incubated at 40 °C for 30 minutes. The reaction was stopped by the addition of 1 ml of 20% (w/v) TCA (at 0 °C) and left in ice bath for 10 min. The mixture was centrifuged and the optical density of the supernatant was mea- sured at 420 nm. One enzyme unit is defined as the amount of enzyme which gives an absorbance increase 0.1 at 420 nm under specified reaction conditions