Protease Activity Assay Using Skim Milk

Proteolytic activity was examined according to the method described by Kessler et al. (2014), Cheng et al. (2022), and Filloux et al. (2014) [18 (link),19 (link),20 (link)] but modified by using skim milk (Skim Milk Powder, Sigma-Aldrich, St. Louis, MO, USA) as a substrate. An overnight Luria Bertani (LB, Becton Dickinson, Holdrege, NE, USA) broth culture was spotted onto a 10% v/v skim milk agar plate and incubated at 37 °C for 24 h. The diameter of the transparent zone under and around the colony was measured and diameters > 5 mm were interpreted as positive for protease activity. The growth of the colony with a clear zone was described as a negative result.