摘要
革兰氏阴性细菌的群体感应系统利用N-酰基高丝氨酸内酯(N-acyl-homoserine lactone,AHL)作为主要信号分子诱导致病因子表达,造成细菌性病害.N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AHLase)能水解AHL分子的内酯键,减弱致病菌的危害.本研究利用从苏云金芽孢杆菌克隆的N-酰基高丝氨酸内酯酶基因(auto inducer inactivation A,aiiA),根据Swiss-model模拟aiiA所编码的AiiA蛋白三维结构,预测可能形成的分子内盐桥、活性中心位点等,利用环状诱变方法对AiiA进行定点突变,以期提高其酶活力和热稳定性等酶学性能.对AiiA及其突变蛋白酶学特性分析结果发现,突变体AiiA-N65K-A206E酶活力要比野生型AiiA-wild提高87.4%,并表现出良好的热稳定性和储存稳定性;37℃温浴30 min后酶活力剩余73.9%,比AiiA-wild有了大幅提高;4℃储存120 h后酶活力剩余12.9%,而AiiA-wild丧失酶活力.酶动力学分析表明,AiiA-N65KA206E酶促反应的米氏常数Km为1.23 mmol/L,与野生型相当;最大反应速率Vmax为32.36μmol/L/min,比野生型有较大提高.本研究表明,利用定点突变技术改造AiiA的分子结构,可有效提升AiiA酶活力、热稳定性和储存稳定性.本研究结果为进一步阐明AiiA结构与功能的关系,促进AiiA在植物病害生物防治上的应用,提供了有益的参考和新的思路.
N-acyl-homoserine lactone （AHL） is a major signaling molecule in quorum sensing （QS） system of Gram-negative bacteria that participates in the interactions between pathogens and plants. Thus, disruption of the QS system of pathogens may inhibit the expression of virulence factors that infect host plants. Recent studies have revealed that many N-acyl-homoserine Lactonase （AHLase） , such as Alia encoded by the Bacillus sp. alia gene, inactivate AHL by hydrolyzing the lactone bond, leading to the disruption of the AHL-mediated QS system. In order to enhance AiiA＇ s enzymatic activity, sitedirected mutagenesis was conducted based on the simulation 3-D structure and active sites of AiiA. The results demonstrate that the mutant AliA- N65K- A206E exhibited the highest enzymatic activity, with 87.4% higher activity than that of the wild type AliA-wild. Thermostability analysis revealed retention of 73.9% enzymatic activity in AliA- N65K-A206E after 30 minute treatment at 37 ℃, as compared to 19.4% enzymatic activity in AiiA-wild. AiiA-N65K-A206E was able to retain 12.9% enzymatic activity after 120 hours under 4 ℃ storage, while AiiA-wild lost its enzymatic activity completely. Kinetic assays revealed that Km value （1.23 mmol/L） in AiiA-N65K-A206E was equivalent to that in wild type, whereas Vmax value （32. 36 μmol/L/min） were higher than that in wild type. Thus the enzymatic activity, thermostability, and storage stability of Alia were all enhanced by site-directed mutagenesis. Our results demonstrate that a relationship between the structure and functions of AiiA and would have an implication in future application of AiiA in plant pathogen bio-control.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2014年第5期453-461,共9页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家高技术研究发展计划(863项目
2011AA10A203)
福建省自然科学基金(No.2012J01123)
福建省教育厅项目(JK2012009)~~
