极端嗜热α-淀粉酶ApkA的高温活性和热稳定性的优化研究

Optimization of the Thermal Activity and Stability of Hyperthermophilic α-amylase ApkA

旨在获得高温活性和热稳定性提高的α-淀粉酶。通过向α-淀粉酶Apk A中引入目前已知的最稳定的α-淀粉酶PFA的Zn^(2+)结合位点,获得Zn^(2+)结合位点突变体ApkAds K152H/A166C。酶学性质分析表明,ApkAds K152H/A166C的高温活性和热稳定性明显提高,最适反应温度由90℃提高至100℃,对应的酶比活力为5 201.08 U/mg。ApkAds K152H/A166C于90℃的半衰期由5 h延长至10 h,于100℃的半衰期由7.5 min延长至80 min。重组α-淀粉酶中Zn^(2+)含量测定结果显示ApkAds K152H/A166C结合了一个Zn^(2+)。结果表明,向ApkA中引入Zn^(2+)结合位点有利于提高其高温活性和热稳定性。

This work aims to obtain α-amylase with improved thermal activity and stability. Based on the structure analysis ofhyperthermophilic α-amylase Apk A and the most thermo-stable α-amylase PFA,a Zn^（2＋）-binding site mutant Apk Ads K152H/A166 C wasconstructed by introducing the Zn^（2＋）-binding site of PFA into Apk A. The mutant Apk Ads K152H/A166 C exhibited effective increase in terms ofthermal activity and stability. The optimal temperature of the mutant increased from 90℃ to 100℃ and the corresponding specific activity was 5201.08 U/mg. The half-life of Apk Ads K152H/A166 C prolonged from 5 h to 10 h while incubated at 90℃,and from 7.5 min to 80 min at 100℃.The results of Zn^（2＋） content measurement in recombinant α-amylases confirmed that Apk Ads K152H/A166 C bound with one Zn^（2＋） ion. Theseresults suggested that the introduction of Zn^（2＋）-binding site improved the thermal activity and stability of Apk A.