Rational Design of Maltogenic Amylase with Enhanced Thermostability Based on Molecular Dynamics Simulation and Its...

Maltogenic amylase (AmyM) hydrolyzes starch to produce maltodextrin, thereby retarding bread staling via starch retrogradation inhibition. However, poor thermal stability limits its industrial application in high-temperature baking. In this study, we employed a computer-aided dual strategy to enhance the thermostability of AmyM-M2 (D261G/T288P) from Bacillus stearothermophilus. Flexible regions were identified via AlphaFold3 and Gromacs molecular dynamics (MD) simulations. By integrating MD-guided saturation and virtual screening-assisted mutagenesis, we obtained two highly stable mutants: M2-A138P and M2-F188I. At 70 °C, their half-lives extended dramatically from 3 h (parent) to 18 and 36 h, respectively. Baking experiments demonstrated both mutants exhibited superior performance in maintaining bread elasticity and delaying hardening compared to the parent enzyme. MD analysis revealed these mutations improve overall structural stability by restricting thermal fluctuations via enhanced local hydrophobic interactions and conformational rigidity. This rational design strategy provides a feasible scheme for engineering the thermostability of industrial enzymes.