Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134

Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH2)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH2 was supplied by an Escherichia coli flavin reductase. TcpC produced in E. coli oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.     

Efficient degradation of 2,4,6-Trichlorophenol requires a set of catabolic genes related to tcp genes from Ralstonia eutropha JMP134(pJP4)

2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are known to degrade this compound. One of these, Ralstonia eutropha JMP134(pJP4), a well-known, versatile chloroaromatic compound degrader, is able to grow in 2,4,6-TCP by converting it to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and beta-ketoadipate. Three enzyme activities encoded by tcp genes, 2,4,6-TCP monooxygenase (tcpA), 6-chlorohydroxyquinol 1,2-dioxygenase (tcpC), and maleylacetate reductase (tcpD), are involved in this catabolic pathway. Here we provide evidence that all these tcp genes are clustered in the R. eutropha JMP134(pJP4) chromosome, forming the putative catabolic operon tcpRXABCYD. We studied the presence of tcp-like gene sequences in several other 2,4,6-TCP-degrading bacterial strains and found two types of strains. One type includes strains belonging to the Ralstonia genus and possessing a set of tcp-like genes, which efficiently degrade 2,4,6-TCP and therefore grow in liquid cultures containing this chlorophenol as a sole carbon source. The other type includes strains belonging to the genera Pseudomonas, Sphingomonas, or Sphingopixis, which do not have tcp-like gene sequences and degrade this pollutant less efficiently and which therefore grow only as small colonies on plates with 2,4,6-TCP. Other than strain JMP134, none of the bacterial strains whose genomes have been sequenced possesses a full set of tcp-like gene sequences.     

Efficient Degradation of 2,4,6-Trichlorophenol Requires a Set of Catabolic Genes Related to tcp Genes from Ralstonia eutropha JMP134(pJP4)

2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are known to degrade this compound. One of these, Ralstonia eutropha JMP134(pJP4), a well-known, versatile chloroaromatic compound degrader, is able to grow in 2,4,6-TCP by converting it to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and β-ketoadipate. Three enzyme activities encoded by tcp genes, 2,4,6-TCP monooxygenase (tcpA), 6-chlorohydroxyquinol 1,2-dioxygenase (tcpC), and maleylacetate reductase (tcpD), are involved in this catabolic pathway. Here we provide evidence that all these tcp genes are clustered in the R. eutropha JMP134(pJP4) chromosome, forming the putative catabolic operon tcpRXABCYD. We studied the presence of tcp-like gene sequences in several other 2,4,6-TCP-degrading bacterial strains and found two types of strains. One type includes strains belonging to the Ralstonia genus and possessing a set of tcp-like genes, which efficiently degrade 2,4,6-TCP and therefore grow in liquid cultures containing this chlorophenol as a sole carbon source. The other type includes strains belonging to the genera Pseudomonas, Sphingomonas, or Sphingopixis, which do not have tcp-like gene sequences and degrade this pollutant less efficiently and which therefore grow only as small colonies on plates with 2,4,6-TCP. Other than strain JMP134, none of the bacterial strains whose genomes have been sequenced possesses a full set of tcp-like gene sequences.     

Conversion of polychlorophenols by laccases with 1-hydroxybenzotriazole as a mediator

The action of purified laccase from the basidial fungi Cerrena unicolor and Trametes sp. on 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) was studied, including reactions involving I-hydroxybenzotriazole as a mediator. Oxidation of 2,4,6-TCP by laccase without the mediator yielded 2,6-dichlorobenzoquinone as a primary conversion product, whereas PCP was not oxidized. Products of further conversion of 2,4,6-TCP and PCP formed with the presence of the mediator.     

Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723.

The biochemistry of pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723 has been studied, and two enzymes responsible for the conversion of PCP to 2,6-dichloro-p-hydroquinone (2,6-DiCH) have previously been purified and characterized. In this study, enzymatic activities consuming 2,6-DiCH were identified from the cell extracts of strain ATCC 39723. The enzyme was purified to apparent homogeneity by a purification scheme consisting of seven steps. Gel filtration chromatography showed a native molecular weight of about 40,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 42,500 Da. The purified enzyme converted 2,6-DiCH to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene), which was easily oxidized by molecular oxygen and hard to detect. The end product, 6-chlorohydroxyquinol, was detected only in the presence of a reductase and NADH in the reaction mixture. The enzyme dechlorinated 2,6-DiCH but not 2,5-DiCH. The enzyme required Fe2+ for activity and was severely inhibited by metal chelating agents. The optimal conditions for activity were pH 7.0 and 40 degrees C. The Kcat for 2,6-DiCH was 35 microM, and the kcat was 0.011 s-1.     

Conversion of polychlorophenols by laccases with 1-hydroxybenzotriazole as a mediator

The action of purified laccase from the basidial fungi Cerrena unicolor and Trametes sp. on 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) was studied, including reactions involving 1-hydroxybenzotriazole as a mediator. Oxidation of 2,4,6-TCP by laccase without the mediator yielded 2,6-dichlorobenzoquinone as a primary conversion product, whereas PCP was not oxidized. Products of further conversion of 2,4,6-TCP and PCP formed with the presence of the mediator.     

Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxidoreductase (TftC) of Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH(2))-utilizing monooxygenase, and FADH(2) was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH(2) and retarded its rapid oxidation by O(2). A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH(2)-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.     

Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100.

Burkholderia cepacia strain AC1100 can be induced for the degradation of 2,4,5-trichlorophenol (2,4,5-TCP). We have purified the active enzyme 30-fold to apparent homogeneity with a 44% yield by a two-step chromatographic procedure, and showed that it consists of a single type of subunit of 59 kDa based on SDS-PAGE using Coomassie blue and Sypro staining. This enzyme has no bound prosthetic group but requires exogenous addition of FAD and NADH to perform the dioxygen-dependent hydroxylation in the 4-position of 2,4,6-TCP. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of dioxygen per mol of 2,4,6-TCP with identification of the reaction product as 2,6-dichlorohydroquinone. Steady state kinetic parameters for cofactors and a variety of substrates were determined. Low K(m) values of 1+/-0.1 microM, 32+/-5 microM and 4+/-2 microM were found for FAD, NADH and 2,6-dichlorophenol (2,6-DCP), respectively, under saturating conditions for the two others. In the presence of 2,6-DCP as a substrate, methimazole (MMI) inhibited the enzyme competitively with a K(i)=27 microM. When other polychlorinated substrates were studied, IC(50) values for MMI were found in a range compatible with their apparent affinity. On the basis of aromatic product formation, NADH and O(2) consumption schemes for 2,4,6-TCP and 2,4,5-TCP degradation are discussed. A Blast search revealed that this enzyme has a high sequence identity (60%) with 2,4,6-TCP-4-monooxygenases from Burkholderia pickettii and from Azotobacter sp. strain GP1 which all of them catalyze para hydroxylative dehalogenation.     

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4 degrees C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors. A combination of these properties of immobilized laccase resulted in the ability to oxidize 2,4,6-trichlorophenol (2,4,6-TCP) at 50 degrees C at pH 7.0. The reactions of soluble and immobilized laccase with 2,4,6-TCP were examined in the presence and absence of redox mediators. 3,5-Dichlorocatechol, 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-1,4-hydroquinone were found to be the primary products of 2,4,6-TCP oxidation by laccase; oligo- and polymeric compounds were also found.     

Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols.

Three strains of Pseudomonas pickettii that can grow with 2,4,6-trichlorophenol (2,4,6-TCP) as the sole source of carbon and energy were isolated from different mixed cultures of soil bacterial populations that had been acclimatized to 2,4,6-TCP. These strains released 3 mol of chloride ion from 1 mol of 2,4,6-TCP during the complete degradation of the TCP. Of these strains, P. pickettii DTP0602 in high-cell-density suspension cultures dechlorinated various chlorophenols (CPs). Cells that were preincubated with 2,4,6-TCP converted isomers of 4-CP to the corresponding chloro-p-hydroquinones, but those preincubated with 4-CP converted CPs lacking a chlorine atom(s) at the o position to isomers of chlorocatechol. The ability of DTP0602 to dechlorinate 2,4,6-TCP was induced by 2,6-dichlorophenol, 2,3,6- and 2,4,6-TCP, and 2,3,4,6-tetrachlorophenol and was repressed in the presence of succinate or glucose.     

Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols.

Three strains of Pseudomonas pickettii that can grow with 2,4,6-trichlorophenol (2,4,6-TCP) as the sole source of carbon and energy were isolated from different mixed cultures of soil bacterial populations that had been acclimatized to 2,4,6-TCP. These strains released 3 mol of chloride ion from 1 mol of 2,4,6-TCP during the complete degradation of the TCP. Of these strains, P. pickettii DTP0602 in high-cell-density suspension cultures dechlorinated various chlorophenols (CPs). Cells that were preincubated with 2,4,6-TCP converted isomers of 4-CP to the corresponding chloro-p-hydroquinones, but those preincubated with 4-CP converted CPs lacking a chlorine atom(s) at the o position to isomers of chlorocatechol. The ability of DTP0602 to dechlorinate 2,4,6-TCP was induced by 2,6-dichlorophenol, 2,3,6- and 2,4,6-TCP, and 2,3,4,6-tetrachlorophenol and was repressed in the presence of succinate or glucose.     

Oxidative 4-dechlorination of 2,4,6-trichlorophenol catalyzed by horseradish peroxidase

 The well-known and easily available horseradish peroxidase (HRP) catalyzes the H₂O₂-dependent oxidative 4-dechlorination of the pollutant 2,4,6-trichlorophenol, which is recalcitrant to many organisms except those producing ligninases. UV-visible spectroscopy and gas chromatography-mass spectrometry identified the oxidized reaction product as 2,6-dichloro-1,4-benzoquinone. NMR and IR spectroscopic data further supported the above characterization. Experimental evidence for the elimination of HCl from the substrate was acquired by detecting the decrease in pH of the reaction mixture, and by observing the presence of the β-chlorocyclopentadienone cation fragment in the mass spectrum of 2,6-dichloro-1,4-benzoquinone. Consequently, nucleophilic attack by water on the 2,4,6-trichlorocyclohexadienone cation was proposed to give the final product. Our results indicate an oxidative dechlorination pathway catalyzed by HRP for 2,4,6-trichlorophenol, similar to that by extracellular lignin peroxidases. The relative catalytic efficiency of HRP seems higher than that of lignin peroxidases. The HRP-H₂O₂ catalytic system could be utilized in the degradation of polychlorinated phenols for industrial and biotechnological purposes. Received: 20 November 1998 / Accepted: 29 January 1999     

Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriavidus necator JMP134

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH₂)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH₂, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (tcpX and tcpB) were cloned, overexpressed, and purified in Escherichia coli. TcpX was purified as a C-terminal His tag fusion (TcpX_H) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpX_H had no activity toward NADPH or riboflavin. Coupling of TcpX_H and TcpA demonstrated that TcpX_H provided FADH₂ for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpX_H in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.     

Purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase from Streptomyces rochei 303: comparison with an analogous enzyme from Azotobacter sp. strain GP1.

The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiol-cleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (kcat/Km = 1.2 and 0.57 s-1.microM-1, respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH2-terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.     

Biodegradation of 2,4,6-tribromophenol by Ochrobactrum sp. strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 microM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

Biodegradation of 2,4,6-Tribromophenol by Ochrobactrum sp. Strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 μM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

Purification and Characterization of Hydroxyquinol 1,2-Dioxygenase from Azotobacter sp. Strain GP1

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bacterium Azotobacter sp. strain GP1 grown with 2,4,6-trichlorophenol as the sole source of carbon. The presumable function of this dioxygenase enzyme in the degradative pathway of 2,4,6-trichlorophenol is discussed. The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) and was found to perform ortho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With the conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 mol of O(inf2) and the formation of 1 mol of chloromaleylacetate were observed. Catechol was not accepted as a substrate. The enzyme has to be induced, and no activity was found in cells grown on succinate. The molecular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4.32. The subunit molecular weight of 34,250 indicates a dimeric structure of the dioxygenase enzyme. The addition of Fe(sup2+) ions significantly activated enzyme activity, and metal-chelating agents inhibited it. Electron paramagnetic resonance data are consistent with high-spin iron(III) in a rhombic environment. The NH(inf2)-terminal amino acid sequence was determined for up to 40 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases.     

The ligninase of Phanerochaete chrysosporium generates cation radicals from methoxybenzenes

The hemoprotein ligninase of Phanerochaete chrysosporium catalyzes, in the presence of H2O2, a variety of seemingly different oxidations in lignin and lignin model compounds. Here we show that the enzyme also catalyzes the oxidation of various methoxybenzenes. ESR spectroscopy shows that the compounds are oxidized to aryl cation radicals. These decompose, evidently by H2O addition. Thus, 1,4-dimethoxybenzene is converted to p-benzoquinone and methanol. We propose a unified mechanism, based on formation of aryl cation radicals, to explain the various reactions catalyzed by the ligninase.     

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2, 4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2, 6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2, 4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor

The toxicity of thirteen isomers of mono-, di-, tri- and pentachlorophenols was tested in potato-dextrose agar cultures of the white rot fungi Panus tigrinus and Coriolus versicolor. 2,4,6-Trichlorophenol (2,4,6-TCP) was chosen for further study of its toxicity and transformation in liquid cultures of these fungi. Two schemes of 2,4,6-TCP addition were tested to minimize its toxic effect to fungal cultures: stepwise addition from the moment of inoculation and single addition after five days of growth. In both cases the ligninolytic enzyme systems of both fungi were found to be responsible for 2,4,6-TCP transformation. 2,6-Dichloro-1,4-hydroquinol and 2,6-dichloro-1,4-benzoquinone were found as products of primary oxidation of 2,4,6-TCP by intact fungal cultures and purified ligninolytic enzymes, Mn-peroxidases and laccases of both fungi. However, primary attack of 2,4,6-TCP in P. tigrinus culture was conducted mainly by Mn-peroxidase, while in C. versicolor it was catalyzed predominantly by laccase, suggesting a different mode of regulation of these enzymes in the two fungi.