Abstract
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.
Highlights
The widespread distribution of aromatic compounds in the environment has led to an increase in pollution, which affects the health quality of living organisms [1]
For the production of C12O, 5 L medium was inoculated with 10% culture inoculum and 600 ppm phenol was added as an inducer
The results showed that C12O activity may be due to homogentisate 1,2-dioxygenase (H12D) expressed in Pseudomonas chlororaphis strain UFB2 (PcUFB2)
Summary
The widespread distribution of aromatic compounds in the environment has led to an increase in pollution, which affects the health quality of living organisms [1]. During the aerobic biodegradation of aromatic compounds, phenol, and it derivatives (e.g., 2,4-dichlorophenoxacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP)), catechols are formed as the central intermediates by the introduction of hydroxyl groups facilitated at ortho- or meta-positions [4,5,6]. The catechol is oxidized via an ortho-cleavage pathway by catechol 1,2-dioxygenase (C12O), or via a meta-pathway to 2-hydroxymuconic semialdehyde by catechol 2,3-dioxygenase (C23O) to open the ring. The enzyme incorporates an oxygen atom into the catechol, resulting in the formation of cis-cis-muconic acid [12,13]. C12O contains Iron(III) oxide as a prosthetic group, and it is part of the enzymes that cleave catechol via the ortho-cleavage, resulting in the formation of cis-cis-muconic acid [14]
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