Industrial-grade α-amylase production, purification, and functional characterization <i>via</i> co-culturing of <i>Aspergillus niger</i> and <i>Aspergillus oryzae</i>

Microbial amylases play an important role in several industrial processes, yet improving production efficiency and enzyme stability remains an ongoing challenge. The present study evaluates a co-cultivation approach using <i>Aspergillus niger</i> and <i>Aspergillus oryzae</i> to enhance α-amylase production under solid-state fermentation using rice husk as an economical substrate. Compared to monoculture systems, co-cultivation resulted in consistently higher enzyme production under all optimized conditions, suggesting a synergistic interaction between the two fungi. Optimal production was achieved at pH 5.0, 30 °C, and 72 hr of incubation, with maximum activity observed at a substrate concentration of 1.5% and 60 °C. The produced α-amylase exhibited stability over a broad range of pH and temperature conditions, retaining 95.84% activity at pH 6.0 after 3 hr and 45.5% activity after 4 hr at 80 °C. Activity was enhanced by Ca²⁺, Mg²⁺, and Cu²⁺ ions, while EDTA caused partial inhibition, indicating metal ion involvement in catalysis. The enzyme also showed tolerance to organic solvents, although detergent exposure reduced activity at higher concentrations. Overall, the results suggest that co-cultivation can improve α-amylase production while yielding an enzyme with properties relevant for industrial use.