Modification of Flexible Regions for Enhanced Thermal Stability of Alkaline Amylase

Alkali-stable amylases offer potential for integrating textile desizing and scouring processes. To meet industrial requirements, molecular engineering strategies were employed to enhance oxidative stability and catalytic efficiency of alkaline amylase. In this study, alkali-stable amylase Amy I from Bacillus halodurans was engineered by modifying multiple highly flexible regions. The sequential truncation of HFR I's N-terminal 40 residues yielded mutant T-40 with 77% higher residual activity than Amy I after incubation at 70 °C for 1 h. Saturation mutagenesis of HFR II/III generated mutant M4, showing 38.9% activity enhancement. CBM-25 substitution in HFR IV further increased activity by 7%. Finally, the integrated mutant Amy I-ML demonstrated exceptional performance with 14.5-fold higher specific activity and 29.6-fold extended half-life (29.4 min at 100 °C) compared to wild-type Amy I. These engineered features-combining thermostability reinforcement with catalytic optimization-establish Amy I-ML as a promising biocatalyst for industrial textile processing. The multiregion engineering approach provided a strategic framework for developing robust industrial enzymes through rational flexibility modulation.