来自Bacillus circulans WZ-12的二氯甲烷脱卤酶基因dcmR的克隆和表达  

Gene Cloning and Overexpression of Dichloromethane Dehalogenase from Bacillus circulans WZ-12

作　　者：吴石金[1] 张华星[2] 胡志航[1] 陈建孟[1] 

机构地区：[1]浙江工业大学生物与环境工程学院,杭州310032 [2]浙江大学宁波理工学院,宁波315100

出　　处：《环境科学》2009年第8期2479-2484,共6页Environmental Science

基　　金：国家自然科学基金项目(20276070);国家高技术研究发展计划(863)项目(2006AA06A310);浙江省环境工程重中之重学科开放基金项目(20080208)

摘　　要：通过PCR方法从Bacillus circulans WZ-12中分离到二氯甲烷脱卤酶基因dcmR,构建具T7强启动子的pET28b（＋）-dcmR质粒,电击转化Escherichia.coli BL21（DE3）,构建了二氯甲烷生物降解基因工程菌BL21[pET28b（＋）-dcmR].重组菌经IPTG诱导后,表达蛋白占总蛋白的32%.表达蛋白的酶活最高可达25.78 U/mL,酶的比活（以蛋白计）为88.86 U/mg.重组菌周质中酶活2.92 U/mL,胞内酶活22.86 U/mL.重组菌产生的酶的活力和比活较原降解菌株高1-2倍.对工程菌的生长特性和降解特性的研究表明,工程菌在LB培养基中的生长特性与原始菌株没有差别,生长至对数期A600 nm值都可达2.4左右.重组菌株dcmR-1在25 h的降解率达90%以上,降解效率比原降解菌株有明显提高.该基因工程菌的构建对二氯甲烷生物降解机制研究以及复合污染环境的生物修复和工程应用具有实际意义.The dcmR gene encoding dichloromethane dehalogenase was amplified by PCR from Bacillus circulans WZ-12 and cloned to expression vector pET28b（ ＋ ）, yielding recombinant plasmid pET28b（ ＋ ）-dcmR. Then plasmid pET28b（ ＋ ）-dcmR was introduced into Escherichia. coli BL21 （DE3）.Expression was induced by IPTG,and the enzyme activity reached 25.78 U/mL, the specifie enzyme activity reached 88.86 U/mg protein. The periplasmic and cytoplasmic enzyme activity reached 2.92 U/mL and 22.86 U/mL respectively. All results analysis demonstrated that the E. coli. strain carrying the dcmR gene could produce dichloremethane dehalogenase efficiently. The growth characteristics of dcmR-1 was compared with the original strain, and the result showed that there was no difference, A600nm of dcmR-1 in LB medium could reach about 2.4 in logarithmic period, which was the same as that of the original strain. The recombinant strain dcm R-1 showed the higher degrading ability than Bacillus circulans WZ-12 and with more than 90% removal efficiency of 120 mmol/L CH2Cl2 in 25 h. All these results indicated that recombinant strain dcmR-1 was a promising strain in bioremediation of CH2Cl2 contaminated environment.

关 键 词：二氯甲烷 脱卤酶基因dcmR 基因克隆与表达 生物降解 

分 类 号：X172[环境科学与工程—环境科学]