Novel Insights for α-amylase Improvement: Leveraging Amylopectin into Accurate Molecular Docking and Mutant Selection

α-Amylase is widely used in various industries. However, current industrial production of α-amylase does not meet the demand, necessitating further enzyme optimization to enhance enzymatic activity. Traditional molecular docking studies have used amylose as a substrate, neglecting the dominant amylopectin structure in natural starch. This oversight may lead to incomplete interaction analyses and inaccurate predictions. This study aimed to investigate the interaction patterns between amylose and amylopectin substrates with α-amylase. To achieve this, we conducted a comprehensive molecular docking analysis. Nine distinct starch structures, comprising linear and branched forms, were docked with α-amylase to explore interaction patterns. Alanine scanning was used to identify key residues within a 3 Å radius of the substrate ligands. Subsequently, saturation mutagenesis was performed on critical residues, followed by experimental validation to assess their impact on enzyme activity. The results revealed that amylopectin substrates provided more accurate predictions of enzyme–substrate interactions. Overall, two mutant α-amylases─AMYI589F and AMYSL335F from Bacillus velezensis and Bacillus licheniformis, respectively─demonstrated α-amylase activity improvements of 101.6 and 89.7%, respectively, compared to their wild-type counterparts. This study underscores the importance of using amylopectin for precise molecular docking analyses, providing a new strategy for optimizing α-amylase and advancing industrial enzyme applications.