Abstract:The development of a thermostable α-amylase for use in starch liquefaction processes is urgently needed. Hyperthermophilic α-amylase possesses good activity at high temperature and thermal stability, and the study of the mechanism underlying the thermal adaptation of hyperthermophilic α-amylase could provide a theoretical basis and design concept for the construction of other thermally stable α-amylases. Based on the amino acid sequence of the α-amylase ApkA produced by the hyperthermophilic archaeon Thermococcus kodakarensis KOD1, a Ca2+-binding site mutant termed ApkAdsN110A/D155A/D164A was constructed. Enzymatic assays suggested that compared to wild-type ApkA, the mutant exhibited significantly reduced thermal activity and stability. The optimal reaction temperature of ApkA was 90 ℃ and the corresponding specific activity was 2946.75 U/mg, while the mutant had an optimal reaction temperature of 80°C with corresponding specific activity of 917.07 U/mg. The half-lives of ApkA and the mutant at 90 ℃ were 5 h and 2 h, respectively. The results suggest that the Ca2+-binding site plays an important role in the maintenance of thermal activity and stability of ApkA, and that Ala substitutions at Asn110, Asp155, and Asp164 lower the thermal activity and stability.
