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Abstract Stability under high‐temperature environments is crucial for amylase to function in starch‐based industries. This study develops a method of modifying protein surfaces by combining computer‐aided tools including FoldX, PoPMuSiC, Discovery Studio, and I‐Mutant 2.0 with conserved sequence analysis. A truncated α‐amylase ∆Amy PTG from Parageobacillus thermoglucosidasius DSMZ 2542 is rationally designed through this method to improve its thermostability. Seven single‐site variants are constructed and five of them displayed enhanced thermostability. Next, three double‐site variants are constructed with one particularly successful double‐site variant N31RT213R, exhibiting a 4.3‐fold longer half‐life at 80 °C. Notably, the specific activity of N31RT213R reaches 10 567.16 U mg −1 , higher than ∆Amy PTG (6645.43 U mg −1 ). When applied to the corn starch liquefaction reaction, the mutant N31RT213R gets a higher yield of product concentration of about 255.70 µg mL −1 , compared to 190.72 µg mL −1 for ∆Amy PTG . Intramolecular forces analysis and surface electrostatic charges analysis are conducted to determine possible causes for the improvement. Also, molecular dynamics simulation is used to analyze the flexibility shifts of the entire protein. This innovative rational engineering approach has proven to be a successful strategy for the selection of hot spots for protein thermostability evolution and has the potential to be applied to other enzymes.
Zhai et al. (Wed,) studied this question.