Influence of Fermentation Strategy on the Production and Functional Properties of Amylases from Bacillus pacificus

Abstract

Amylase is an important industrial enzyme widely applied across food, pharmaceutical, and biotechnology sectors. Bacillus species are preferred producers because of their robustness, high secretion capacity, and metabolic versatility. However, conventional production systems have not kept pace with increasing global demand, prompting the need for more efficient bioprocessing strategies. This study reports the purification and characterization of amylase from Bacillus pacificus cultivated using submerged fermentation (SmF) and electrofermentation (EF). Purification was carried out through cold acetone precipitation followed by Sephadex G-100 gel filtration. The resulting enzymes were evaluated for temperature and pH optima, stability, and metal ion responsiveness. SmF-derived amylase showed optimal activity of 41.4 U/mL at 50 °C and retained 75% activity after 3 hours at this temperature. In contrast, EF amylase displayed higher optimal activity of 47.2 U/mL at 60 °C, maintaining 99% activity after 3 hours. Both enzymes exhibited maximum activity at pH 7.0, recording 40.94 U/mL (SmF) and 41.73 U/mL (EF). Metal ion assays indicated that Li⁺ and Na⁺ enhanced EF enzyme activity but inhibited the SmF enzyme at higher concentrations, while Co²⁺ increased activity in both systems, with a more pronounced effect in SmF. Kinetic analysis further showed superior catalytic performance in the EF enzyme, with a Vmax of 46.49 U/mL and a Km of 0.001835 mM compared to 46.42 U/mL and 0.0058 mM for the SmF enzyme. Overall, EF amylase exhibited improved thermal and pH stability, higher catalytic efficiency, and stronger substrate affinity. These findings highlight electrofermentation as a promising and scalable alternative for industrial amylase production.