Brewer’s spent grain valorization by Yarrowia lipolytica for protease production: Process optimization and enzyme characterization

The valorization of agro-industrial waste as raw material for bioprocesses represents a fundamental strategy for advancing circular economy approaches. In this study, malt bagasse (MB), the main byproduct of the brewing industry, was evaluated as a substrate to produce extracellular protease by Yarrowia lipolytica in submerged fermentation. The compositional analysis of MB confirmed its suitability as a nutrient-rich lignocellulosic matrix capable of supporting microbial growth and enzymatic secretion. The process optimization, using a central composite rotatable factorial design, identified the MB concentration as the main factor influencing protease production, with a maximum activity of 466.29 U/L obtained at 25 g/L of MB and 0.6 g/L of urea after 24 h, representing a five-fold increase compared to non-optimized conditions. The enzyme showed optimal activity at 25 °C and pH 7, with stability under refrigeration for up to 120 days. Kinetic analysis revealed a K _m of 3.08mg/mL and a V _max of 570.30.72 U/L, indicating high catalytic efficiency. These results demonstrate that BSG can be effectively converted into a high-value-added biocatalyst, reinforcing its potential for integration into sustainable bioprocesses. Thus, the use of BSG not only enables the valorization of the residue but also supports the production of functionally relevant enzymes with potential for industrial applications. Statement of Novelty Brewer’s spent grain is one of the largest by-products of the brewing industry, yet its conversion into high-value bioproducts remains limited. This study demonstrates an integrated strategy to valorize this residue as the main substrate for extracellular protease production by Yarrowia lipolytica . By combining medium optimization, bench-scale cultivation, and biochemical characterization, we show that brewer’s spent grain can support efficient enzyme production while reducing the need for conventional culture media. The optimized process substantially increased protease production and yielded an enzyme with favorable catalytic properties and storage stability. These findings provide a practical waste-to-value approach that supports circular bioeconomy principles and expands the potential industrial applications of brewer’s spent grain beyond its traditional low-value uses.