Whey, a large-scale dairy industry by-product, can be converted into whey protein concentrate (WPC), providing a cost-effective nutrient-rich substrate for microbial fermentation. We investigated protease production by Aspergillus oryzae using WPC as the sole substrate in submerged fermentation. Following fermentation, the protease was purified sequentially from the crude extract by salting-out, which yielded a substantial purification factor (~39), and subsequent ion-exchange chromatography. The non-adsorbed chromatographic fraction showed the highest protease activity (92.6 U/mL) and revealed one main protein band ~45 kDa via SDS-PAGE. Enzyme characterization demonstrated activity across neutral-to-alkaline conditions, optimal at pH 9.0 and 37 °C, with stability maintained between 30 °C and 37 °C. The enzyme was classified as a serine protease based on strong inhibition by PMSF and SDS; its activity was also inhibited by Zn²⁺, Mg²⁺, and K⁺, but enhanced by Ca²⁺. This work validates WPC as an efficient substrate for protease production by A. oryzae and presents a promising strategy for valorizing industrial by-products through sustainable biotechnology.

Proteases are among the most commercially important industrial enzymes, yet their large-scale production is often limited by complex and costly downstream processing. In this study, an integrated bioprocess was developed for the production, in situ recovery, and purification of an extracellular protease produced by Aspergillus sp. (SIS 22/UCP 1287) under submerged fermentation. Enzyme extraction was coupled directly to fermentation using a polyethylene glycol (PEG)–phosphate aqueous two-phase system (ATPS), aiming to enhance recovery while preserving enzymatic activity. The effects of PEG molecular weight, polymer and phosphate concentrations, and pH on enzyme partitioning were systematically investigated through a full factorial experimental design. Low-molecular-weight PEG and near-neutral pH conditions significantly favored enzyme migration to the PEG-rich phase. Under optimized conditions (15% PEG 3500, 20% phosphate, pH 7.0), the ATPS achieved a partition coefficient of 65.55, enzyme recovery of 209%, and a purification factor of 1.64. Subsequent purification by DEAE–Sephadex ion-exchange chromatography yielded a tenfold increase in specific activity, with optimal elution at 0.5 M NaCl. SDS–PAGE analysis confirmed the homogeneity of the purified protease, revealing a single band at approximately 59 kDa. Overall, the proposed integrated ATPS–chromatography strategy represents a robust, scalable, and environmentally friendly platform that significantly simplifies downstream processing while maintaining high enzyme activity, highlighting its potential for industrial and biotechnological applications.