High-performance protease biosynthesis from Aspergillus niger through rice polish valorization and RSM-based optimization

Proteases are widely used industrial biocatalysts, but their economical and sustainable production remains challenging. This study investigates protease production by Aspergillus niger using rice polish as a low-cost substrate under solid-state fermentation. Among six agro-industrial residues evaluated, rice polish supported the highest baseline protease activity (1.63 ± 0.05 U/mL). Preliminary optimization using a one-factor-at-a-time approach identified 3 days of incubation, 37 °C, and pH 6.5 as favorable conditions for enzyme production. Further optimization was performed using response surface methodology by evaluating the combined effects of biomass loading (3–7 g), inoculum size (1.5–3.5 mL), Mg 2+ concentration (0.25–0.55 g), and moisture content (40–60%). The statistical model was significant (R 2 = 0.9873, p < 0.0001) and predicted a maximum protease activity of 396.15 U/mL, which was experimentally validated with an observed value of 392.72 ± 0.14 U/mL under optimized conditions. Partial purification by ammonium sulfate precipitation and dialysis resulted in a 2.8-fold increase in specific activity with 46% recovery. The protease exhibited optimal activity at pH 6.5 and 37 °C, maintaining more than 80% stability between pH 5.5 and 7.0 and up to 40 °C. Application studies demonstrated effective enzymatic dehairing of goat skin within 90 minutes, with improved mechanical properties compared with conventional chemical treatment. Overall, the findings highlight rice polish as a promising substrate for protease production and demonstrate the utility of statistical optimization in improving enzyme yield for potential industrial applications. • Rice polish supported higher protease production than other tested agro-industrial residues. • Combined OFAT and RSM approaches significantly improved protease yield under SSF conditions. • Statistical optimization identified biomass, inoculum, Mg 2+ , and moisture as key variables. • Partial purification enhanced specific activity with acceptable recovery of enzyme activity. • The protease showed effective dehairing performance under mild operating conditions.