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Bacillus amyloliquefaciens α‐amylase (BAA) is one of well‐known midrange thermostability amylases that are widely used in food and washing processes. However, the improvement of its catalytic properties by molecular modification is still lagging. To improve the activity of α‐amylase BAA, mutants BAA28 and BAA294 are constructed via error‐prone PCR and purified by column chromatography in the present study. The catalytic efficiencies (Kcat/Km) of BAA28 and BAA294 are 2.42 and 2.73 mL mg−1 s−1, which are 43% and 61% higher than that of the wild‐type BAA, respectively. Their specific activities are also increased by 40% and 62%, respectively, with no apparent changes of optimum temperature and pH. Homology modeling and molecular docking analysis suggest that the reduced steric hindrance is an important factor that enhances catalytic efficiencies and specific activities of the variants. These results may deepen the understanding of the mechanisms underlying the effects of each mutation on the catalytic efficiency of BAA and facilitate the construction of potent BAA mutants. [ABSTRACT FROM AUTHOR]

The medium-temperature alpha-amylase of Bacillus amyloliquefaciens is widely used in the food and washing process. Enhancing the thermostability of alpha-amylases and investigating the mechanism of stability are important for enzyme industry development. The optimal temperature and pH of the wild-type BAA and mutant MuBAA (D28E/V118A/S187D/K370 N) were all 60 °C and 6.0, respectively. The mutant MuBAA showed better thermostability at 50 °C and 60 °C, with a specific activity of 206.61 U/mg, which was 99.1% greater than that of the wild-type. By analyzing predicted structures, the improving thermostability of the mutant MuBAA was mainly related to enhanced stabilization of a loop region in domain B via more calcium-binding sites and intramolecular interactions around Asp187. Furthermore, additional intramolecular interactions around sites 28 and 370 in domain A were also beneficial for improving thermostability. Additionally, the decrease of steric hindrance at the active cavity increased the specific activity of the mutant MuBAA. Improving the thermostability of BAA has theoretical reference values for the modification of alpha-amylases. • Mutant alpha-amylase MuBAA improved the thermostability and specific activity. • More calcium-binding sites and forces of Asp187 enhanced the stability of a loop region. • Increasing intramolecular interactions made the structure of domain A tighter. • Decreasing the steric hindrance at the active cavity increased the specific activity. [ABSTRACT FROM AUTHOR]