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

摘要:

摘要:【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。

关键词:关键词:N-酰基高丝氨酸内酯，N-酰基高丝氨酸内酯酶，酶学特性，定点突变

Abstract:

Abstract:［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 Nacylhomoserine 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 AiiA (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．

Key words:Keywords: N-acylhomoserine lactone，N-acylhomoserine lactonase，enzymatic characterization，site-directed mutagenesis