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

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.

Key words:Pseudomonas putida ND6; catechol 1; 2-dioxygenase; catA; catechol ortho-cleavage pathway