Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathways, respectively. The aim of this study was to purify, characterize, and predict the template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 (PcUFB2). Preliminary studies showed that PcUFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02% by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and a temperature of 30 °C. The Lineweaver⁻Burk plot showed the vmax and Km values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O shared 81% homology with homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. The characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in PcUFB2 and its possible application in bioremediation strategies.
Keywords: Pseudomonas chlororaphis; Pseudomonas chlororaphis strain UFB2; catechol 1,2-dioxygenase; homogentisate 1,2-dioxygenase.