Abstract: Hyperthermophilic α-amylases, which are active and stable at high temperatures, are of great interest forresearchers studying starch liquefaction. Research into the molecular basis of thermal adaptation of hyperthermophilicα-amylase can provide theoretical guidance to improve the thermal activity and thermal stability of α-amylases. Based onthe sequence analysis of hyperthermophilic α-amylase ApkA from Thermococcus kodakarensis KOD1, a signal peptidedeleted mutant (ApkAds) and an A180K site mutant (ApkAdsA180K) were constructed. Compared with ApkAds, the mutantApkAdsA180K exhibited a sharp increase in thermal activity and stability. The optimal temperature of ApkA was 90 ℃ andthe corresponding specific activity was 2 946.75 U/mg, while the optimal temperature of the mutant was 100 ℃ and thecorresponding specific activity was 4 501.08 U/mg. When incubated at 90 ℃, ApkAds and the mutant exhibited half-livesof 5 h and 7 h, respectively. The tertiary structure of ApkAdsA180K obtained by homologous modeling indicated that K180and D212 are involved in salt bridge formation. These results suggest that the salt bridge between K180 and D212 plays animportant role in maintaining the thermal activity and stability of ApkAdsA180K.

Key words: hyperthermophilic α-amylase, salt bridge, site-directed mutagenesis, thermal activity, thermal stability