2.8. Protease activity assays

The protease fluorescent detection kit (Sigma‐Aldrich) was used for routine detection of proteolytic activity as previously described.32, 34 Briefly, 10 µL lysate or 5 μM enzyme was assessed for activity on FITC‐casein in 50 mM TrisHCl pH 8.5 (at RT), 50 mM NaCl, in absence or presence of 1 mM CaCl₂ in a total volume of 50 μL at 37 °C for 1 h unless otherwise stated. Temperature optimum was assayed using the FITC‐casein assay. For the mutants, activity was assessed using EnzChek™ Protease Assay Kit (ThermoFischer). 10 μg/mL BODIPY FL casein was prepared by resuspending the substrate in 50 mM Tris‐HCl pH 8.5 (at RT) and 50 mM NaCl. 12.5 μL of BODIPY‐FL casein was used per reaction, with 10 μL cleared extract in 50 mM Tris pH 8.5 (at RT), 50 mM NaCl and 1 mM CaCl₂ in a final volume of 100 μL. Samples were incubated at 37 °C for 1 h, and fluorescence was read.

pH optimum was determined using 1 µM Asn3‐ISP, 350 µM N‐succinyl‐AAPF p‐nitroanilide (Sigma‐Aldrich) in 50 mM NaCl, 1 mM CaCl₂, and 50 mM buffer (citrate buffer pH 3.0‐6.0, acetate buffer pH 4.0‐6.0, sodium phosphate buffer pH 6.0‐8.0, Tris‐HCl buffer pH 7.0‐9.0 and glycine buffer pH 9.0‐11.0). Reaction was run at 25 °C for 20 min, in presence of excess substrate.