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The gene of catechol 1, 2-dioxygenase was identified and cloned from the genome of Oceanimonas marisflavi 102-Na3. The protein was expressed in Escherichia coli BL21 (DE3) and purified to homogeneity of a dimer with molecular mass of 69.2 kDa. The enzyme was highly stable in pH 6.0-9.5 and below 45 °C and exhibited the maximum activity at pH 8.0 and 30 °C. Being the first characterized intradiol dioxygenase from marine bacteria Oceanimonas sp., the enzyme showed catalytic activity for catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and pyrogallol. For catechol, K m and V max were 11.2 μM and 13.4 U/mg of protein, respectively. The enzyme also showed resistance to most of the metal ions, surfactants and organic solvents, being a promising biocatalyst for biodegradation of aromatic compounds in complex environments., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

The detection of aromatic compounds from pesticides and industrial wastewater has become of great interest, since these compounds withstand chemical oxidation and biological degradation, accumulating in the environment. In this work, a highly sensitive biosensor for detecting catechol was obtained with the immobilization of Cl-catechol 1,2-dioxygenase (CCD) in nanostructured films. CCD layers were alternated with poly(amidoamine) generation 4 (PAMAM G4) dendrimer using the electrostatic layer-by-layer (LbL) technique. Circular dichroism (CD) measurements indicated that the immobilized CCD preserved the same conformation as in solution. The thickness of the very first CCD layers in the LbL films was estimated at ca. 3.6 nm, as revealed by surface plasmon resonance (SPR). PAMAM/CCD 10-bilayer films were employed in detecting diluted catechol solutions using either an optical or electrical approach. Due to the mild immobilization conditions employed, especially regarding the pH and ionic strength of the dipping solutions, CCD remained active in the films for periods longer than 3 weeks. The optical detection comprised absorption experiments in which the formation of cis-cis muconic acid, resulting from the reaction between CCD and catechol, was monitored by measuring the absorbance at 260 nm after film immersion in catechol solutions. The electrical detection was carried out using LbL films deposited onto gold-interdigitated electrodes immersed in aqueous solutions at different catechol concentrations. Using impedance spectroscopy in a broad frequency range (1Hz-1kHz), we could detect catechol in solutions at concentrations as low as 10(-10) M.