Preprint Article Version 1 This version is not peer-reviewed

Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas Chlororaphis Strain UFB2

Version 1 : Received: 11 October 2018 / Approved: 12 October 2018 / Online: 12 October 2018 (03:45:41 CEST)

How to cite: Setlhare, B.; Kumar, A.; Mokoena, M.P.; Olaniran, A.O. Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas Chlororaphis Strain UFB2. Preprints 2018, 2018100247 (doi: 10.20944/preprints201810.0247.v1). Setlhare, B.; Kumar, A.; Mokoena, M.P.; Olaniran, A.O. Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas Chlororaphis Strain UFB2. Preprints 2018, 2018100247 (doi: 10.20944/preprints201810.0247.v1).

Abstract

Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathway, respectively. The aim of the study was to purify, characterize and predict 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 temperature 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 share 81% homology to homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. 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.

Subject Areas

catechol 1,2-dioxygenase; homogentisate 1,2-dioxygenase; Pseudomonas chlororaphis; Pseudomonas chlororaphis strain UFB2

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