Abstract: In this study, protease was purified consecutively by ammonium sulfate precipitation, Q-HP anion-exchange chromatography and Superdux 75 gel chromatography. Matrix-assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) mass spectrometry analysis suggested that the protease was highly similar to calpain RIM13, a cysteine protease from Aspergillus oryzae. The optimal temperature was 50 ℃ and the optimal pH was 6.5. The enzyme was stable at 40 ℃ and pH 7.0. It was activated by Mn2+, but inhibited by Fe3+, Fe2+, Cu2+, Ca2+, K+ and Na+. The metal ions affected its secondary structure. The Km and Vm values of the protease for casein were 2.43 g/L and 103.09 mg/(L·min), respectively. At NaCl concentrations of 5, 10 and 15 g/100 mL, the protease retained 77.22%, 54.39% and 41.15% of its initial activity, respectively. Consequently, the protease has potential industrial applications.

Key words: Aspergillus oryzae, protease, purification, enzymatic properties