Enhanced Production of Acid Phosphatase in Bacillus subtilis: From Heterologous Expression to Optimized Fermentation Strategy

Acid phosphatases (ACPase, EC 3.1.3.2) are hydrolytic enzymes widely distributed in both plant and animal tissues. Despite their ubiquitous presence, the production and specific activity of ACPase in these sources remain suboptimal. Consequently, the development of microbial cell factories for large-scale ACPase production has emerged as a significant research focus. In this study, we successfully expressed the phosphatase PAP2 family protein (acid phosphatase) from Acinetobacter nosocomialis 1905 in Bacillus subtilis 168. The specific activity of the crude enzyme solution was 59.60 U/mg. After purification, the enzyme activity increased to 86.62 U/mL, with a specific activity of 129.60 U/mg. Characterization of the enzyme revealed optimal activity at 45 °C and a pH of 6.0. The Km value was determined to be 0.25 mmol/L using p-nitrophenylphosphoric acid disodium salt as the substrate. Additionally, the enzyme activity was found to be enhanced by the presence of Ni2+. Dissolved oxygen and medium were subsequently optimized during fermentation on the basis of a commercially available 5 L bioreactor. The recombinant strain B. subtilis 168/pMA5-Acp achieved maximal volumetric enzyme activity of 136.9 U/mL after 12 h of fermentation under optimized conditions: an aeration rate of 1.142 VVM (4 lpm), an agitation speed of 350 rpm, and an optimal ratio of lactose to fish powder (7.5 g/L:12.5 g/L). These optimizations resulted in a 5.9-fold increase in volumetric enzyme activity, a 4.9-fold increase in enzyme synthesis per unit cell volume, and a 48.6% increase in biomass concentration. This study establishes a comprehensive technological framework for prokaryotic fermentation-based ACPase production, potentially addressing the bottleneck in industrial-scale applications.