Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement

α-Amylase (EC 3.2.1.1) from Geobacillus stearothermophilus (generally recognized as safe) exhibited thermal inactivation, hampering its further application in starch-based industries. To address this, we performed structural analyses based on molecular dynamics targeting the flexible regions of α-amylase. Subsequently, we rationally designed a thermostable mutant, AmyS1, by introducing disulfide bonds to stabilize the flexible regions. AmyS1 showed excellent thermostability without any stability-activity trade-off, giving a 40-fold longer T1/2 (1359 min) at 90 °C. Thermostability mechanism analysis revealed that the introduction of disulfide bonds in AmyS1 refined weak spots and reconfigured the protein's force network. Moreover, AmyS1 exhibited improved pH compatibility and enhanced corn starch liquefaction at 100 °C with a 5.1-fold increased product concentration. Baking tests confirmed that AmyS1 enhanced bread quality and extended the shelf life. Therefore, mutant AmyS1 is a robust candidate for the starch-based industry.