Production and biochemical characterization of an Aspergillus oryzae protease preparation using dairy whey under submerged fermentation and evaluation of its milk-clotting activity

Proteases are among the most commercially important industrial enzymes due to their broad applications in food, pharmaceutical, detergent, and biotechnological processes. In this context, bovine dairy whey, a major by-product of the dairy industry, represents a promising low-cost substrate for sustainable enzyme production. This study aimed to produce, purify, and biochemically characterize a protease from Aspergillus oryzae cultivated under submerged fermentation using bovine dairy whey as substrate. Initially, A. aculeatus, A. ochraceus, and A. oryzae were evaluated for proteolytic activity. The selected enzyme was pre-purified by ammonium sulfate precipitation, ion-exchange chromatography, and Superdex G-200 gel filtration, followed by biochemical characterization. Aspergillus oryzae exhibited the highest proteolytic activity (96 U/mL). The enzyme achieved a purification fold of 56.63 and specific activity of 84.38 U/mg. Maximum activity was observed at pH 5.0 and 50 °C, whereas greater thermal stability occurred at 40 °C. PMSF and EDTA strongly inhibited enzymatic activity. The inhibition by PMSF suggests the possible involvement of serine residues in the catalytic activity, thus, consistent with a serine protease. Mn2+ and Fe2+ maintained residual activities above 80%, whereas Cu2+ caused the strongest inhibition. Additionally, the partially purified enzyme showed expressive milk-clotting activity, reaching 75.94 MCU and 91.60% coagulation after gel filtration chromatography. These findings highlight the biotechnological potential of A. oryzae proteases produced from bovine dairy whey for sustainable enzyme production and milk-clotting applications within circular economy approaches.