开发耐高温α-淀粉酶是目前淀粉液化工艺的迫切需要，极端嗜热α-淀粉酶具有优良的高温活性和热稳定性，其高温适应性机制研究可以为构建耐高温α-淀粉酶提供理论依据和设计思路。通过分析来源于极端嗜热古生菌Thermococcus kodakarensis KOD1的α-淀粉酶ApkA的氨基酸序列，构建Ca2+结合位点突变体ApkAdsN110A/D155A/D164A。酶学性质分析表明，与野生型ApkAds相比，突变体ApkAdsN110A/D155A/D164A的高温活性和热稳定性明显降低。其中ApkAds的最适反应温度为90 ℃，对应的绝对酶活为2946.75 U/mg；突变体ApkAdsN110A/D155A/D164A的最适反应温度为8 0℃，对应的绝对酶活为917.07 U/mg。ApkAds于90 ℃的半衰期约为5 h，突变体ApkAdsN110A/D155A/D164A于90 ℃的半衰期约为2 h。本研究结果表明ApkA中Ca2+结合位点与其高温活性和热稳定性均相关，Asn110、Asp155及Asp164这三个氨基酸残基的丙氨酸替换突变不利于ApkA维持其高温活性和热稳定性。

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.

国家自然科学基金资助项目（31501422）；江西省青年科学基金资助项目（20151BAB214001）；江西省科学院资助项目（2014-YYB-08、2014-XTPH1-08）