Precipitation of proteases from enzymatic crude extract

CEs were precipitated with cold acetone (− 20 °C) in a ratio of 1:1 (v/v) or with cold ethanol (− 20 °C) in a ratio of 1:1 (v/v). The solvent was added slowly. The mixtures were kept for 1 h at – 20 °C. Next, the precipitates were separated by decantation and centrifugation at 12,000×g and 4 °C for 15 min. The precipitates were maintained at 5 °C for 16 h to remove the residual solvent and then they were freeze-dried. The freeze-dried samples were stored at – 80 °C for further analysis.