枯草芽孢杆菌SS6N-酰基高丝氨酸内酯酶基因的克隆表达及其酶学特性  

Gene cloning,expression and characterization of Nacylhomoserine lactonase from Bacillus subtilis SS6

作　　者：张盼[1,2] 丁贤[3] 李来好[3] 陆勇军[4] 殷波[1] 

机构地区：[1]中国科学院南海海洋研究所,广东广州510301 [2]中国科学院大学,北京100049 [3]中国水产科学研究院南海水产研究所农业部水产品加工重点实验室,广东广州510300 [4]中山大学生命科学院生物防治国家重点实验室,广东广州510275

出　　处：《微生物学报》2015年第6期739-747,共9页Acta Microbiologica Sinica

基　　金：广东省教育部产学研结合项目(2011B090400151);(中国科学院南海水产研究所)农业部水产品加工重点实验室开放基金(NYJG201308)~~

摘　　要：【目的】从一株具有细菌群体感应(Quorum Sensing,QS)信号分子淬灭活性的枯草芽孢杆菌(Bacillus subtilis)SS6中扩增N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,Aii A)基因aii ASS6并异源表达,研究此信号降解酶的酶学特性。【方法】设计特异性引物,从B.subtilis SS6中克隆N-酰基高丝氨酸内酯酶基因aii ASS6,测序并进行生物信息学分析;将此基因克隆到表达载体p ET28(a),构建重组菌株并提纯目的蛋白Aii ASS6;然后用高效液相色谱(high performance liquid chromatography,HPLC)分析目的蛋白Aii ASS6降解QS信号分子N-(3-Oxooctanoyl)-L-homoserine lactone(OOHL)的酶学特性。【结果】克隆得到基因片段,命名为aii ASS6(Gen Bank:KP125494),其编码一条含有297氨基酸残基的多肽,用p ET28(a)成功构建重组质粒p ET28-aii ASS6。生物信息学分析表明,Aii ASS6的氨基酸序列含有N-酰基高丝氨酸内酯酶典型的"HXHXDH"基序和194位的Tyr残基。在Escherichia coli BL21(DE3)中异源表达Aii ASS6,用Ni柱纯化后,Aii ASS6含量达2.76 mg/m L。HPLC检测结果表明Aii ASS6对OOHL具有很强的催化活性及耐热性,Km和Vmax分别为0.998 mmol/L和22.3 U/mg,最适pH为7.6,最适温度范围为50-90℃;此酶在4℃保存3个月后其残余活性仍达到86%,表现出较强的稳定性。【结论】从B.subtilis SS6中获得的QS淬灭酶Aii ASS6表现出降解QS信号分子的高活性,其酶学特性表明它具有作为微生物制剂防控植物或水产养殖中基于QS调控的病原菌毒力的应用潜力。Objective] This study was aimed to obtain a quorum quenching N-acylhomoserine lactonase gene from Bacillus subtilis SS6 and to characterize the enzyme.[Methods] We amplified N-acylhomoserine lactonase gene from B.subtilis SS6 by PCR methods.Gene aii ASS6 was cloned into the expression vector of p ET28（a） and transformed into Escherichia coli BL21.The activity of Aii ASS6 on signal N-（3-Oxooctanoyl）-L-homoserine lactone was characterized by high performance liquid chromatography（HPLC）.[Results] We successfully cloned an N-acylhomoserine lactonase gene from B.subtilis SS6 strain,namely aii ASS6（Gen Bank： KP125494）.The sequencing result showed that the length of aii ASS6 was 891 bp and the gene contained an Open Reading Frame encoding 297 amino acids.HPLC results showed that Aii ASS6 was very active between 50 and 90℃ and the optimal pH was 7.6.Lineweaver-Burk treatment of the data yielded apparent Kmand the Vmax was 0.998 mmol/L and 22.3 U/mg,respectively.Moreover,the relative activity of the enzyme remained 86% after storing at 4℃ for 3 months.[Conclusion] Our findings will be helpful for further studies.

关 键 词：群体感应 N-酰基高丝氨酸内酯酶 酶学特性 高效液相色谱 

分 类 号：Q78[生物学—分子生物学] Q936