Enhanced Production, Enzymatic Activity, and Thermostability of an α-Amylase from Bacillus amyloliquefaciens in Lactococcus lactis

A novel alpha-amylase gene (BAA) from Bacillus amyloliquefaciens was cloned into Lactococcus lactis, designing two recombinant alpha-amylases to facilitate extracellular secretion. Following optimizing the expression conditions, the highest yield of BAA (88.12 mmol/L) was achieved upon 36 h induction and 5 ng/mL nisin concentration. Determining the enzymatic properties of BAA revealed its poor stability and activity at high temperatures, hindering its widespread application. Therefore, we used computer-aided design to generate a mutant, S275L, which exhibited significantly improved activity and thermostability: an 18.7% increase in enzymatic activity (3767.38 U/mg), a 10 degrees C increase in optimal temperature, and a 49.2% improvement in stability at 60 degrees C. Molecular dynamics simulations and force analysis confirmed these enhancements. Finally, the mutant S275L's potential was further analyzed for starch hydrolysis on poultry feed. Therefore, the mutant S275L holds promising as an enzyme agent for enhanced feed digestibility and quality.