A rapid method for naphtalene dioxygenase assay in whole cells of naphtalene cis-dihydrodiol dehydrogenase blocked Pseudomonas fluoresecens: Screening of potential inducers of dioxygenase activity

Cidaria, D.; Deidda, Federica; Bosetti, Aldo

doi:10.1007/bf00167286

Cited by 18 publications

(10 citation statements)

References 13 publications

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“…Therefore, we chose the mutant strain TTC1 in a 3-liter flask. To ensure the standardization of the culture conditions, we also used the enzyme activity test previously reported (12). When the yield was very low, we used either the E. coli recombinant pVL1343 ϩ pMS13 (10) or the reactor procedure (13) to get enough product to characterize.…”

Section: Resultsmentioning

confidence: 99%

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Specificity of Substrate Recognition by Pseudomonas fluorescens N3 Dioxygenase

Gennaro

Sello

Bianchi

et al. 1997

Journal of Biological Chemistry

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Pseudomonas fluorescens N3 is able to grow on naphthalene as the sole carbon and energy source. The mutant TTC1, blocked at the dihydrodiol dehydrogenase level, which can transform the hydrocarbon into the corresponding dihydrodiol, has been used to produce bioconversion products. To rationalize the different grades of conversion obtained with different substrates, a study was performed using non-naphthalene derivatives, including benzenes, conjugated benzenes, and polycyclic aromatic hydrocarbons. The corresponding diols obtained by bioconversion have been isolated and characterized. A theoretical model that considers both energy and geometry factors has been proposed to rationalize the experimental data. Good agreement has been found between the calculated values and the experimental results.Pseudomonas dioxygenases are a family of closely related enzymes that can add an oxygen molecule to a substrate double bond (1). Their ability to make such a transformation on aromatic compounds, often the first step in the biodegradation of such compounds (2), is particularly interesting. The high stability of aromatic compounds requires an unusually high redox potential that, in most cases, is made available by two or three component enzymes (3). The active site is believed to belong to the class of Rieske-type iron-sulfur proteins, in which the iron, coordinated by two histidine nitrogens and two cysteine sulfurs, is, at the same time, the end point of a long redox chain transporting the necessary electrons from NADH (or NADPH) and the coordination site of molecular oxygen (3). In the general framework of this type of dioxygenase, the specificity of recognition could be related to the geometric and functional characteristics of the active site. As a consequence, there are well-known oxygenases that transform monocyclic compounds (benzene (4) or toluene (5)), naphthalenes (6), or polycyclic aromatic hydrocarbons (7).Since 1992, our research group has been interested in exploiting the power of the dioxygenase of P. fluorescens N3 that we isolated from the activated sludge of a wastewater treatment plant (8). The wild type is able to completely degrade naphthalene and some of its derivatives and to transform many other naphthalenes into the corresponding salicylic acids (8).We later isolated a mutant strain (TTC1) blocked at the dihydrodiol dehydrogenase level (9), and then we cloned the naphthalene dioxygenase gene in Escherichia coli JM109 (10). Thus, we could efficiently produce dihydrodiols from many naphthalenes carrying substituents in both position 1 and position 2. The yield seemed to be correlated to two aspects, the electronic characteristics of the substituents and their position, and was higher for electron releasing groups in position 2. However, the yield trend was not readily understandable, so we decided to extend our investigation to assess the specificity of N3 dioxygenase.Two strongly correlated actions were considered: the experimental verification of substrate specificity (by testing diverse aromatic s...

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Section: Resultsmentioning

confidence: 99%

“…Naphthalene Dioxygenase Assay-The naphthalene dioxygenase activity was determined by a photometric method as described previously (12). One unit of dioxygenase activity was defined as the amount of cells (dry cell weight) producing 1 mol of 1,2-dihydro-1,2-dihydroxynaphthalene in 1 min under the test conditions.…”

Section: Methodsmentioning

confidence: 99%

Specificity of Substrate Recognition by Pseudomonas fluorescens N3 Dioxygenase

Gennaro

Sello

Bianchi

et al. 1997

Journal of Biological Chemistry

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“…The first step in degradation of naphthalene (the derivative of which is naproxen) is the hydroxylation of C1 and C2 catalysed by naphthalene dioxygenase [46]. It was suggested that this enzyme is responsible for hydroxylation of C7 and C8 of naproxen.…”

Section: The Influence Of Immobilization On Enzymes Activitymentioning

confidence: 99%

“…In order to determine monooxygenase activity (with phenol or naproxen as a substrate), NADH oxidation (ε 340 = 6220/M cm) was measured spectrophotometrically [58]. The naphthalene dioxygenase, gentisate 1,2-dioxygenase and salicylate 1,2-dioxygenase activity was measured spectrophotometrically by the formation of cis,cis-dihydrodiol (ε 262 = 8230/M cm) [46], maleylpyruvate (ε 330 = 10,800/M cm) [49] and 2-oxohepta-3,5-dienedioic acid (ε 283 = 13,600/M cm) [48], respectively. Protein concentration was determined using the Bradford method [59].…”

Section: Enzyme Assaymentioning

confidence: 99%

Immobilization of Planococcus sp. S5 Strain on the Loofah Sponge and Its Application in Naproxen Removal

et al. 2018

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Planococcus sp. S5, a Gram-positive bacterium isolated from the activated sludge is known to degrade naproxen in the presence of an additional carbon source. Due to the possible toxicity of naproxen and intermediates of its degradation, the whole cells of S5 strain were immobilized onto loofah sponge. The immobilized cells degraded 6, 9, 12 or 15 mg/L of naproxen faster than the free cells. Planococcus sp. cells immobilized onto the loofah sponge were able to degrade naproxen efficiently for 55 days without significant damage and disintegration of the carrier. Analysis of the activity of enzymes involved in naproxen degradation showed that stabilization of S5 cells in exopolysaccharide (EPS) resulted in a significant increase of their activity. Changes in the structure of biofilm formed on the loofah sponge cubes during degradation of naproxen were observed. Developed biocatalyst system showed high resistance to naproxen and its intermediates and degraded higher concentrations of the drug in comparison to the free cells.

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“…The strains were grown for 36 h at 30³C. The preparation of the cell suspension and all measurements were performed by using the method for the determination of naphthalene dioxygenase activity in whole cells [16].…”

Section: Naphthalene Dioxygenase Assaymentioning

confidence: 99%

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida

Mordukhova

2000

FEMS Microbiology Letters

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Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation. The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied. Indole-3-acetic acid synthesis by these transconjugants was 15^30 times as much in contrast to a wild-type strain with glucose as the sole carbon source. No difference was observed in other carbon or nitrogen sources. It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid. ß

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A rapid method for naphtalene dioxygenase assay in whole cells of naphtalene cis-dihydrodiol dehydrogenase blocked Pseudomonas fluoresecens: Screening of potential inducers of dioxygenase activity

Cited by 18 publications

References 13 publications

Specificity of Substrate Recognition by Pseudomonas fluorescens N3 Dioxygenase

Specificity of Substrate Recognition by Pseudomonas fluorescens N3 Dioxygenase

Immobilization of Planococcus sp. S5 Strain on the Loofah Sponge and Its Application in Naproxen Removal

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida

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