Ca^2+结合位点对极端嗜热α-淀粉酶ApkA高温活性及热稳定性的影响

Effect of the Ca^(2+)-binding Site on Thermal Activity and Stability of the Thermococcus kodakarensis Hyperthermophilic α-Amylase Apk A

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Apk A的氨基酸序列,构建Ca2+结合位点突变体Apk Ads N110A/D155A/D164A。酶学性质分析表明,与野生型Apk Ads相比,突变体Apk Ads N110A/D155A/D164A的高温活性和热稳定性明显降低。其中Apk Ads的最适反应温度为90℃,对应的绝对酶活为2946.75 U/mg;突变体Apk Ads N110A/D155A/D164A的最适反应温度为80℃,对应的绝对酶活为917.07 U/mg。Apk Ads于90℃的半衰期约为5 h,突变体Apk Ads N110A/D155A/D164A于90℃的半衰期约为2 h。本研究结果表明Apk A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Apk A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 Apk A produced by the hyperthermophilic archaeon Thermococcus kodakarensis KOD1,a Ca^2＋-binding site mutant termed Apk Ads N110A/D155A/D164 A was constructed.Enzymatic assays suggested that compared to wild-type Apk A,the mutant exhibited significantly reduced thermal activity and stability.The optimal reaction temperature of Apk A 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 Apk A and the mutant at 90 ℃ were 5 h and 2 h,respectively.The results suggest that the Ca^2＋-binding site plays an important role in the maintenance of thermal activity and stability of Apk A,and that Ala substitutions at Asn110,Asp155,and Asp164 lower the thermal activity and stability.