【摘要】 恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的Km为0.019μmol/L,Vmax为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe2+对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。更多还原

【Abstract】 Catechol 1,2-dioxygenase gene,catA,from naphthalene-degrading plasmid pND6-1 of Pseudomonas putida ND6,was cloned and expressed in Escherichia coli.Enzymic properties of the expressed product were investigated.The results indicated that the K_m and V_maxof the enzyme are 0.019 mol/L and 1.434 mol/（min·mg）,respectively.The enzyme possessed a thermal stability and 93.7% activity was retained after incubating at 50℃ for 45min.Fe²⁺could enhance the enzyme activity by 292%.The enzyme displayed a lower activity against 4-chlorocatechol and belongs to group Ⅰ of catechol 1,2-dioxygenases.When naphthalene was used as a substrate for growth of strain ND6,catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were both detected in their crude extract.However,when strain ND6 was grown on benzoate,ρ-hydroxybenzoic acid or phenylacetic acid as a sole source of carbon the activity of catechol 1,2-dioxygenase was much higher than that of catechol 2,3-dioxygenase.These indicated that strain ND6 is able to metabolize naphthalene by catechol meta-and ortho-cleavage pathways.When benzoate,ρ-hydroxybenzoic acid and phenylacetic acid were used as growth substrates,strain ND6 degrades these compounds only by catechol ortho-cleavage pathway.更多还原