定点突变提高N-酰基高丝氨酸内酯酶酶活和温度稳定性

Enhancement of enzyme activity and thermostability of Nacylhomoserine lactonase by site-directed mutagenesis

【目的】提高N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AiiA)酶活及温度稳定性。【方法】本研究基于AiiA同源蛋白的三维结构对AiiA进行定点突变,分析野生型AiiA及其突变蛋白酶活和温度稳定性。【结果】野生型AiiA较不稳定,在45℃下温浴30 min,或4℃储存5 d后均失去降解N-酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL)的活性。但是突变AiiA蛋白(N65K,T195R和A206E)的酶活力较野生型AiiA均提高了20%以上,且4℃储存时间延长到7 d。此外,突变株N65K比野生型AiiA对高温具有更强的耐受性,在45℃温浴后剩余酶活力达到45%以上,55℃温浴30 min后仍保留5.0%的酶活力。【结论】通过定点突变改造AiiA蛋白结构,提升了AiiA蛋白的酶活和温度稳定性。

[ Objective ] To enhance enzymatic activity and thermostability of N-acylhomoserine lactonase （AiiA）. [Methods] We performed site-directed mutagenesis based on AiiA homologous 3-D protein structure, and analyzed enzymatic activity and thermostability of both wild type and mutated AiiA. [ Results] The wild type AiiA lost its N- acylhomoserine lactone （AHL） degrading activity after being incubated at 45℃ for 30 min or after being stored at 4℃ for 5 days. By comparison, the AHL-degrading activities of three types of mutated Alia （N65K, T195R, and A206E） were enhanced, and their storage periods at 4℃ were extended to 7 days. In addition, the N65K mutant acquired higher temperature tolerance with remain of more than 45% of its enzymatic activity after being incubated at 45℃ and 5.0% enzymatic activity after being incubated at 55℃ as compared to the wild type. [ Conclusion] Molecular modulation by site-directed mutagenesis could significantly improve enzymatic activity and thermostability of AiiA.