Zhao Hua-bing, Chen Wei, Cai Bao-li
College of Life Sciences, Nankai University, Tianjin 300071, China.
Wei Sheng Wu Xue Bao. 2007 Jun;47(3):387-91.
Catechol 1,2-dioxygenase gene, calA, 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 Km and Vmax of the enzyme are 0.019 mol/L and 1.434 mol/(min x mg), respectively. The enzyme possessed a thermal stability and 93.7% activity was retained after incubating at 50 degrees C for 45 min. 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, rho-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, rho-hydroxybenzoic acid and phenylacetic acid were used as growth substrates, strain ND6 degrades these compounds only by catechol ortho-cleavage pathway.
从恶臭假单胞菌ND6的萘降解质粒pND6-1中克隆了儿茶酚1,2-双加氧酶基因calA，并在大肠杆菌中进行表达。对表达产物的酶学性质进行了研究。结果表明，该酶的Km和Vmax分别为0.019 mol/L和1.434 mol/(min·mg)。该酶具有热稳定性，在50℃孵育45 min后仍保留93.7%的活性。Fe2+可使酶活性提高292%。该酶对4-氯儿茶酚的活性较低，属于儿茶酚1,2-双加氧酶的I组。当萘用作菌株ND6生长的底物时，在其粗提物中检测到儿茶酚1,2-双加氧酶和儿茶酚2,3-双加氧酶的活性。然而，当菌株ND6以苯甲酸、对羟基苯甲酸或苯乙酸作为唯一碳源生长时，儿茶酚1,2-双加氧酶的活性远高于儿茶酚2,3-双加氧酶的活性。这些表明菌株ND6能够通过儿茶酚间位和邻位裂解途径代谢萘。当苯甲酸、对羟基苯甲酸和苯乙酸用作生长底物时，菌株ND6仅通过儿茶酚邻位裂解途径降解这些化合物。
