Simultaneous Improvement of Acid Tolerance and Thermostability of Bacillus acidicola α-Amylase via Semi-Rational Design and Its Application in the Production of Resistant Dextrin

Abstract

Thermostable and acid-tolerant α-amylases are highly desirable for efficient starch processing and resistant dextrin production. Here, an α-amylase from Bacillus acidicola (Ba-amy) was engineered to simultaneously improve the thermostability and acid tolerance by semirational design. Residues 178–179 in a protruding loop were deleted, a 37-residue N-terminal fragment from acid-tolerant α-amylase was introduced and surface residue Val332 was substituted with Asp. The resulting mutant Mut-V332D exhibited a 14.0-fold increase in specific activity (6734.8 U/mg), improved acid tolerance at pH 5.5, and a 5.8-fold longer half-life at 90 °C (85.5 min) compared with Ba-amy. Mechanistic analysis suggested that enhanced thermostability resulted from reduced loop flexibility, strengthened hydrogen-bond interactions and optimized surface electrostatics, while the N-terminal extension contributed to acid tolerance. Furthermore, Mut-V332D demonstrated potential for resistant dextrin production without repeated pH adjustment. This work provides an effective strategy for engineering robust α-amylases for industrial applications.