Abstract: This work aims to investigate the effect of Ca²⁺-binding site which locates between the domain A and the raw starch binding domain of thermoacidiphilic raw starch degrading α-amylase GTamy on the enzymatic properties of GTamy. A Ca²⁺-binding site mutant GTamyD407A/D430A was constructed based on the strucutre analysis of GTamy. Results showed that:Compared to GTamy,the mutant exhibited significant decrease in terms of thermal activity and stability. The specific activity of the mutant at 80℃ decreased from 1 756.75 U/mg to 1 484.48 U/mg,and the half-life of the mutant at 80℃ decreased from 3 h to 2.5 h. The mutant showed similar substrate binding capacity but 79% of the catalytic efficiency of GTamy on the soluble starch. And the mutant exhibited 67.9% of the substrate binding rate and 59.3% of the hydrolysis rate of GTamy towards corn starch. The Ca²⁺-binding site might influence the thermal activity and stability of GTamy by changing the molecular structure of the raw starch binding domain and the catalytic active center. This result indicated that the Ca²⁺-binding site was important to the thermal activity and stability of GTamy.