恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

Cloning and expression of catA gene from Pseudomonas putida ND6 and study on the catechol cleavage pathway

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K_m为0.019μmol/L,V_max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。

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_max of 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.