Optimization of Mutation Sites in Bacillus Velezensis Serine Protease for Enhanced Acid and Thermal Stability in Animal Feed Applications

Serine proteases from Bacillus species are widely used in the feed industry, but their poor tolerance to high acidity and temperature limits their digestive efficacy in animals. To overcome this, we engineered the serine protease SprD1 from Bacillus velezensis D1 via structure-guided site-directed mutagenesis, using protein folding free energy (ΔΔ G ) analysis and loop flexibility modification. Multiple single and five combinatorial mutants were designed, among which the double mutant Mut5 showed the highest improvements. Mut5 retained 60 kU/mL activity at pH 2.0, about 3.3-fold higher than wild-type SprD1 (18 kU/mL), and maintained 36.4 kU/mL after 12 h at pH 2.2, demonstrating outstanding acid resistance. It preserved an ∼10% increase in activity after 4 h at 50–60 °C. In simulated gastric digestion of animal feed, Mut5 significantly enhanced amino acid release, with enzyme dosage and substrate concentration identified as key efficiency determinants. Overall, Mut5 is a high-performance protease combining acid stability, heat resistance, and superior feed hydrolysis.