Biodegradation of 3-nitrotoluene by Rhodococcus sp. strain ZWL3NT

A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.     

Oxidative release of nitrite from 2-nitrotoluene by a three-component enzyme system from Pseudomonas sp. strain JS42.

Pseudomonas sp. strain JS42 utilizes 2-nitrotoluene (2NT) as the sole source of carbon and energy for growth. Intact cells catalyze the oxidation of 2NT to 3-methylcatechol and nitrite in a reaction that requires molecular oxygen. Cell extracts oxidized 2NT to 3-methylcatechol and nitrite in the presence of NAD(P)H and ferrous iron. Ion-exchange chromatography yielded three protein fractions (A, B, and C) which were all required for the oxidation of 2NT to 3-methylcatechol and nitrite. Component B (reductase2NT) catalyzed a NAD(P)H-dependent reduction of cytochrome c. Solutions of component A (ISP2NT) were brown and showed absorption maxima at 458 and 324 nm. Two major bands with M(r)s 52,500 and 28,000 were observed when ISP2NT was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component C could be replaced by ferredoxin NAP from the Pseudomonas putida NCIB 9816-4 naphthalene dioxygenase system and was given the designation ferredoxin2NT. Experiments with 18O2 showed that both oxygen atoms were added to the aromatic ring of 2NT to yield 3-methylcatechol. The enzyme is a new multicomponent enzyme system which we have designated 2NT 2,3-dioxygenase.     

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.     

Biomineralization of 3-nitrotoluene by Diaphorobacter species

Three bacterial strains utilizing 3-nitrotoluene (3-NT) as a sole source of carbon, nitrogen and energy were isolated from an industrial wastewater treatment plant. Biochemical tests and 16S rDNA sequence analysis revealed that the isolated strains belonged to Diaphorobacter sp. Detailed studies were carried out with Diaphorobacter sp. strain DS2. Degradation of 3-NT by Diaphorobacter sp. strain DS2 was accompanied by the release of nitrite in the culture broth with increase in biomass. Total organic carbon analysis confirmed the extensive mineralization of 3-NT. The strain could degrade 3-methylcatechol, 4-methylcatechol and catechol easily suggesting that the degradation pathway could involve these as possible intermediates. Successful PCR amplification of the oxygenase large subunit and the presence of high activity for catechol 2,3-dioxygenase in the crude cell lysate further confirmed that the degradation of 3-NT occurred through (methyl)catechol intermediates in strain DS2. The strain DS2 was found to degrade other isomers of mononitrotoluene (2-NT and 4-NT) and nitrobenzene as well.     

Biodegradation of 2-Nitrotoluene by <Emphasis Type="Italic">Micrococcus</Emphasis> sp. strain SMN-1

A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-nitrotoluene through 3-methylcatechol by a meta-cleavage pathway, with release of nitrite.     

Microbial degradation of alkylbenzenesulphonates. Metabolism of homologues of short alkyl-chain length by an Alcaligenes sp

1. An organism isolated from sewage and identified as an Alcaligenes sp. utilized benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate as sole source of carbon and energy for growth. Higher alkylbenzenesulphonate homologues and the hydrocarbons, benzene, toluene, phenylethane and 1-phenyldodecane were not utilized. 2. 2-Phenylpropanesulphonate was metabolized to 4-isopropylcatechol. 3. 1-Phenylpropanesulphonate was metabolized to an ortho-diol, which was tentatively identified, in the absence of an authentic specimen, as 4-n-propylcatechol. 4. In the presence of 4-isopropylcatechol, which inhibited catechol 2,3-dioxygenase, 4-ethylcatechol accumulated in cultures growing on phenylethane-p-sulphonate. 5. Authentic samples of catechol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol and 3-isopropylcatechol were oxidized by heat-treated extracts to the corresponding 2-hydroxyalkylmuconic semialdehydes. Ring cleavage occurred between C-2 and C-3. 6. The catechol derived from 1-phenylpropanesulphonate was oxygenated by catechol 2,3-dioxygenase to a compound with all the properties of a 2-hydroxyalkylmuconic semialdehyde, but it was not rigorously identified. 7. The catechol 2,3-dioxygenase induced by growth on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate showed a constant ratio of specific activities with catechol, 3-methylcatechol, 4-methylcatechol and 4-ethylcatechol that was independent of the growth substrate. At 60°C, activity towards these substrates declined at an identical first-order rate. 8. Enzymes of the `ortho' pathway of catechol metabolism were present in small amounts in cells grown on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate. 9. The catechol 1,2-dioxygenase oxidized the alkylcatechols, but the rates and the total extents of oxidation were less than for catechol itself. The oxidation products of these alkylcatechols were not further metabolized.     

Metabolism of phenol and cresols by Bacillus stearothermophilus.

An obligate thermophilic strain of Bacillus stearothermophilus, strain PH24, isolated from industrial sediment by elective culture, grew readily at 55 C on phenol or on one of the isomers of cresol as the major carbon source. Intact cells grown in the presence of phenol, o-cresol, m-cresol, or p-cresol were induced to oxidize, without lag, these substrates together with catechol, 3-methylcatechol, and 4-methylcatechol. Cell extracts prepared from B. stearothermophilus PH24 after growth in the presence of phenol converted phenol to catechol with a concomitant uptake of 1 mol of oxygen per mol of substrate in reaction mixtures supplemented with reduced nicotinamide adenine dinucleotide. These preparations also catalyzed the oxidation of o-cresol to 3-methylcatechol and of m-cresol and p-cresol to 4-methylcatechol. Enzyme activity was inhibited by 1 mM p-chloromercuribenzoate and by 0.1 mM 0-phenanthroline. Catechol and the corresponding methylcatechol intermediates were further dissimilated by cell extracts of phenol-grown cells via the meta-cleavage route to yield 2-hydroxymuconic semialdehyde and the respective methylated derivatives.     

The metabolism of cresols by species of Pseudomonas

1. A comparison of rates of oxidation of various compounds by whole cells indicated that protocatechuate was a reaction intermediate when a non-fluorescent species of Pseudomonas oxidized p-cresol. In contrast, a fluorescent Pseudomonas oxidized 3-methylcatechol and 4-methylcatechol when grown with p-cresol, but did not oxidize protocatechuate. 2. Heat-treated extracts of the fluorescent Pseudomonas oxidized catechol, 3-methylcatechol and 4-methylcatechol to ring-fission products, the spectroscopic properties of which were recorded. Rates of enzymic degradation of these products were also measured. 3. Acetic acid and formic acid were obtained by the action of a Sephadex-treated extract on 3-methylcatechol and 4-methylcatechol respectively. In each case 0.8mol. of the carboxylic acid was formed from 1.0mol. of substrate. 4. Dialysed extracts converted 3-methylcatechol into acetaldehyde and pyruvate, with 4-hydroxy-2-oxovalerate as a reaction intermediate. 4-Methylcatechol was converted first into 4-hydroxy-2-oxohexanoate and then into propionaldehyde and pyruvate. 5. The ring-fission product of catechol was formed from phenol by a fluorescent Pseudomonas, that of 3-methylcatechol was formed from o-cresol and m-cresol, and the ring-fission product of 4-methylcatechol was given from p-cresol. Propionate was readily oxidized by these cells after growth with p-cresol, but this compound was not attacked when phenol, o-cresol or m-cresol served as source of carbon. 6. Cell extracts appeared to attack only one enantiomer of synthetic 4-hydroxy-2-oxohexanoate.     

Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol.

Partially purified preparations of catechol 2,3-dioxygenase from toluene-grown cells of Pseudomonas putida catalyzed the stoichiometric oxidation of 3-methylcatechol to 2-hydroxy-6-oxohepta-2,4-dienoate. Other substrates oxidized by the enzyme preparation were catechol, 4-methylcatechol, and 4-fluorocatechol. The apparent Michaelis constants for 3-methylcatechol and catechol were 10.6 and 22.0 muM, respectively. Substitution at the 4-position decreases the affinity and activity of the enzyme for the substrate. Catechol 2,3-dioxygenase preparations did not oxidize 3-chlorocatechol. In addition, incubation of the enzyme with 3-chlorocatechol led to inactivation of the enzyme. Kinetic analyses revealed that both 3-chlorocatechol and 4-chlorocatechol were noncompetitive or mixed-type inhibitors of the enzyme. 3-Chlorocatechol (Ki = 0.14 muM) was a more potent inhibitor than 4-chlorocatechol (Ki = 50 muM). The effect of the ion-chelating agents Tiron and o-phenanthrolene were compared with that of 3-chlorocatechol on the inactivation of the enzyme. Each inhibitor appeared to remove iron from the enzyme, since inactive enzyme preparations could be fully reactivated by treatment with ferrous iron and a reducing agent.     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain.

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth.

Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition ratio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4ECR6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Molecular oxygen was required for the inactivation of the wild-type enzyme by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactivated enzymes could be reactivated by incubation with FeSO4 plus ascorbic acid. The enzyme inactivation is thus most likely mechanism based and occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols lower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.     

Characterization of catechol 2,3-dioxygenases.

Three catechol 2,3-dioxygenases for biphenyl, naphthalene/salicylate, and toluene/xylene oxidation were cloned from Achromobacter xylosoxidans KF701, Pseudomonas putida (NAH7), and Pseudomonas sp. (pWWO). The cloned catechol 2,3-dioxygenases were identified by enzymatic activity assay in addition to yellow bands on polyacrylamide gel after electrophoresis and activity staining. All of the cloned catechol 2,3-dioxygenases exhibited their highest activities on catechol as a substrate compared with catechol derivatives including 4-chlorocatechol, 3-methylcatechol, and 4-methylcatechol. The cloned catechol 2,3-dioxygenases are not fused proteins but were significantly different from one another in their electrophoretic mobilities on nondenaturing 7.5%-polyacrylamide gel.     

Evolution of a new bacterial pathway for 4-nitrotoluene degradation

Bacteria that assimilate synthetic nitroarene compounds represent unique evolutionary models, as their metabolic pathways are in the process of adaptation and optimization for the consumption of these toxic chemicals. We used Acidovorax sp. strain JS42, which is capable of growth on nitrobenzene and 2-nitrotoluene, in experiments to examine how a nitroarene degradation pathway evolves when its host strain is challenged with direct selective pressure to assimilate non-native substrates. Although the same enzyme that initiates the degradation of nitrobenzene and 2-nitrotoluene also oxidizes 4-nitrotoluene to 4-methylcatechol, which is a growth substrate for JS42, the strain is incapable of growth on 4-nitrotoluene. Using long-term laboratory evolution experiments, we obtained JS42 mutants that gained the ability to grow on 4-nitrotoluene via a new degradation pathway. The underlying basis for this new activity resulted from the accumulation of specific mutations in the gene encoding the dioxygenase that catalyses the initial oxidation of nitroarene substrates, but at positions distal to the active site and previously unknown to affect activity in this or related enzymes. We constructed additional mutant dioxygenases to identify the order of mutations that led to the improved enzymes. Biochemical analyses revealed a defined, step-wise pathway for the evolution of the improved dioxygenases.     

Oxidative Pathway for the Biodegradation of Nitrobenzene by Comamonas sp. Strain JS765

Previous studies have shown that the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 proceeds by the reduction of nitrobenzene through nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage. We report here the isolation of a Comamonas sp. that uses an oxidative pathway for the complete mineralization of nitrobenzene. The isolate, designated strain JS765, uses nitrobenzene as a sole source of carbon, nitrogen, and energy. Nitrobenzene-grown cells oxidized nitrobenzene, with the stoichiometric release of nitrite. Extracts of nitrobenzene-grown JS765 showed high levels of catechol 2,3-dioxygenase activity that were not abolished by heating the cell extracts to 60(deg)C for 10 min. The ring cleavage product had an absorbance maximum at 375 nm, consistent with that of 2-hydroxymuconic semialdehyde. Both NAD-dependent dehydrogenase and NAD-independent hydrolase activities towards 2-hydroxymuconic semialdehyde were induced in extracts of nitrobenzene-grown cells. Catechol accumulated in the reaction mixture when cells preincubated with 3-chlorocatechol were incubated with nitrobenzene. Conversion of nitrobenzene to catechol by induced cells in the presence of 3-chlorocatechol and (sup18)O(inf2) demonstrated the simultaneous incorporation of two atoms of oxygen, which indicated that the initial reaction was dioxygenation. The results indicate that the catabolic pathway involves an initial dioxygenase attack on nitrobenzene with the release of nitrite and formation of catechol, which is subsequently degraded by a meta cleavage pathway.     

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6.

Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3-dihydroxy-1-methylbenzene (3-chloro-6-methylcatechol), 4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (p-CT dihydrodiol), and 2-methyl-4-carboxymethylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.     

Metabolism of naphthalene, 2-methylnaphthalene, salicylate, and benzoate by Pseudomonas PG: regulation of tangential pathways.

Naphthalene is metabolized by Pseudomonas PG through 1,2-dihydroxynaphthalene and salicylate to catechol, which is then degraded by the meta pathway. 2-Methylnaphthalene, but not 1-methylnaphthalene, also serves as a growth substrate and is metabolized by the same route, through 4-methylcatechol. The same nonspecific meta pathway enzymes appear to be induced by growth on either naphthalene or 2-methylnaphthalene. The level to which 2-hydroxymuconic semialdehyde hydrolase is induced is low and probably of no metabolic significance. Growth on salicylate or catechol, both intermediates of naphthalene degradation, or benzoate results in induction of the ortho pathway, the alternative route for catechol dissimilation. No induction of 1,2-dihydroxynaphthalene oxygenase was found in salicylate-grown cells. Anaerobic growth on a succinate-nitrate medium in the presence of various inducers indicates that cis, cis-muconate, or one of its metabolites is the inducer of the ortho pathway enzymes. The inducer or inducers of the early enzymes of naphthalene degradation and of the meta pathway enzymes must be an early intermediate of the naphthalene pathway above salicylate.     

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6

Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3-dihydroxy-1-methylbenzene (3-chloro-6-methylcatechol), 4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (p-CT dihydrodiol), and 2-methyl-4-carboxymethylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.     

Some kinetic properties of polyphenol oxidase from Thymbra spicata L. var. spicata

Polyphenol oxidase (PPO) of Thymbra (Thymbra spicata L. var. spicata) was isolated by (NH₄)₂SO₄ precipitation and dialysis. A diphenolase from Thymbra plant, active against 4-methylcatechol, catechol and pyrogallol was characterized in detail in terms of pH and temperature optima, stability, kinetic parameters and inhibition behaviour towards some general PPO inhibitors. 4-Methylcatechol was the most suitable substrate, due to the lowest K_m and the biggest V_max/K_m values, followed by catechol and pyrogallol. The Thymbra PPO had maximum activity at pH 5.0, 7.0 and 8.0 with 4-methylcatechol, catechol and pyrogallol substrates, respectively. The optimum temperature of activity for Thymbra PPO was 30, 40 and 50 °C for 4-methylcatechol, catechol and pyrogallol substrates, respectively. It was found that optimum temperature and pH were substrate-dependent studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time. Inhibition of Thymbra PPO was investigated with inhibitors such as l-cysteine and glutathione using 4-methylcatechol, catechol and pyrogallol as substrates. It was found that l-cysteine was a more effective inhibitor than glutathione owing to lower K_i. The type of inhibition depended on the origin of the PPO studied and also on the substrate used. Furthermore, the IC₅₀ values of inhibitors sudied on PPO were determined by means of activity percentage (I) diagrams.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.