红球菌35R1中邻苯二酚1,2-双加氧酶的表达及纯化  

Expression and purification of catechol 1,2-dioxygenase from Rhodococcus sp.35R1

作　　者：何航航 姚霓红 吴菁 闫达中[1] 晁红军 HE Hang-hang;YAO Ni-hong;WU Jing;YAN Da-zhong;CHAO Hong-jun(School of Life Science and Technology,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]武汉轻工大学生命科学与技术学院,武汉430023

出　　处：《武汉轻工大学学报》2021年第3期20-23,72,共5页Journal of Wuhan Polytechnic University

摘　　要：3,5-二甲基苯酚(3,5-DMP)是一种重要的精细化工原料以及有机合成中间体,在工农业生产领域广泛应用并大量排放到环境中,对人类和环境造成了巨大的危害。Rhodococcus sp.35R1是一株以3,5-二甲基苯酚为唯一碳源和能源的高效降解菌。为验证菌株35R1中3,5-二甲基苯酚是通过邻苯二酚途径开环代谢的,通过生物信息学分析,寻找到35R1基因组上的邻苯二酚1,2-双加氧酶编码基因catA,通过一步克隆法将其连接到表达载体pET28a(+)上,然后,将其转化至表达宿主E.coli BL21(DE3)中,从而进行该基因的异源表达。结果成功在35R1基因组上扩增到catA基因,构建了重组质粒pET28a-catA,采用自诱导的方法诱导表达了邻苯二酚1,2-双加氧酶,纯化酶可催化邻苯二酚生成顺式粘糠酸。3,5-dimethylphenol(3,5-DMP) is an important fine chemical raw material and organic synthesis intermediate, which is widely used in industrial and agricultural production.Rhodococcus sp.35 R1 was grown on 3,5-dimethylphenol as the sole source of carbon and energy.In order to verify that 3,5-dimethylphenol is metabolized by the catechol ring-cleavage pathway in strain 35 R1,the catA gene encoded catechol 1,2-dioxygenase on the genome of strain 35 R1 was cloned into pET28 a(+) through One Step Cloning method and heterologously expressed in E.coli BL21(DE3).Results: The catA gene was successfully amplified in 35 R1 genome, and constructed recombinant pET28 a-catA.The recombinant pET28 a-catA was transformed into E.coli BL21(DE3),which was induced by the auto-induced-media.The catechol 1,2-dioxygenase CatA was further purified and determined to catalyze catechol to muconate.

关 键 词：3 5-二甲基苯酚 邻苯二酚 邻苯二酚1 2-双加氧酶 基因克隆 异源表达 

分 类 号：Q78[生物学—分子生物学]