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PubMedDate not supplied

Water purification through bioconversion of phenol compounds by tyrosinase and chemical adsorption by chitosan beads.

Enzymatic removal of various phenol compounds from artificial wastewater was undertaken by the combined use of mushroom tyrosinase (EC 1.14.18.1) and chitosan beads as function of pH value, temperature, tyrosinase dose, and hydrogen peroxide-to-substrate ratio. Chitosan film incubated in a p-crersol+tyrosinase mixture had the main peaks at 400-470 nm assigned to chemically adsorbed quinone derivatives, which increased over the immersion time. These results indicate that removal of phenol compounds is caused by their tyrosinase-catalyzed oxidation to the corresponding quinone derivatives and the subsequent chemical adsorption on the chitosan film. The optimum conditions for quinone adsorption were determined to be pH 7 and 45 degrees C for p-cresol. Some alkyl-substituted phenol compounds were removed by adsorption of quinone derivatives enzymatically generated on the chitosan beads, and the % removal for p-cresol, 4-ethylphenol, 4-n-propylphenol, 4-n-butylphenol, and p-chlorophenol went up to 93%. In addition, 4-tert-butylphenol underwent tyrosinase-catalyzed oxidation in the presence of hydrogen peroxide. This procedure was applicable to removal of chlorophenols and alkyl-substituted phenols.

Adsorption↗
PubMed2001

Mechanistic aspects of the de-novo synthesis of PCDD/PCDF on model mixtures and MSWI fly ashes using amorphous 12C- and 13C-labeled carbon.

The formation of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) from amorphous 12C- and 13C-labeled carbon was studied on model mixtures and real fly ashes. PCDD/F can either be formed directly (de-novo) from carbon already present in fly ash or step-by-step via condensation of two aromatic rings. Using model mixtures containing 12C- and 13C-labeled carbon in various ratios we observed the formation of the following compound classes: 12C6-PCPh, -PCBz, 13C6-PCPh, -PCBz, 12C12-PCDD/ F, 13C12-PCDD/F, and 12C6 13C6-PCDD/F. By examining the fraction of the mixed PCDD/F (one of the two aromatic ring is composed solely of 12C-atoms while the other contains only 13C-atoms) in the total concentration of PCDD/F, conclusions on the formation of these three ring structures are possible. From the experimental results, it can be concluded that both reaction mechanisms are operative in the formation of PCDD/F from carbon. On fly ashes approximately half of the total amount of PCDD is formed via condensation of de-novo created C6-precursors e.g. chlorophenols, while the remainder is directly released (de-novo) from the carbon i.e., formed from a related C12-structure. However, the condensation of intermediate aromatic C6-precursors is of minor importance in the formation of PCDF. With increasing temperature the relative amount of the 12C6 13C6-PCDD formed by condensation decreases due to the faster evaporation of chlorophenols. At a constant reaction temperature, the ratio of both reaction pathways is hardly influenced by reaction time. In experiments with fly ashes doped with 13C-labeled carbon, this carbon isotope shows a similar reactivity as the native carbon present on the fly ash. Thus, the used amorphous carbons are suitable models for this investigation.

Benzofurans↗
PubMed2005

Formation of chloroform and chlorinated organics by free-chlorine-mediated oxidation of triclosan.

The widely used antimicrobial agent triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) readily reacts with free chlorine under drinking water treatment conditions. Overall second-order kinetics were observed, first-order in free chlorine and first-order in triclosan. Over the pH range of 4-11.5, the kinetics were pH sensitive as a result of the pH dependent speciation of both triclosan and free chlorine. Using a Marquardt-Levenberg routine, it was determined that this pH effect indicates that the dominant reaction in this system is between the ionized phenolate form of triclosan and hypochlorous acid (HOCl). The overall second-order rate coefficient was determined to be kArO- = 5.40 (+/- 1.82) x 10(3) M(-1) s(-1). Three chlorophenoxyphenols and two chlorophenols were identified by gas chromatographic-mass spectroscopic analysis. The chlorophenoxyphenol compounds include two monochlorinated triclosan derivatives (5,6-dichloro-2-(2,4-dichlorophenoy)phenol and 4,5-dichloro-2-(2,4-dichlorophenoxy)phenol) and one dichlorinated derivative (4,5,6-trichloro-(2,4-dichlorophenoxy)phenol); these species form via bimolecular electrophilic substitution of triclosan. 2,4-Dichlorophenol was detected under all reaction conditions and forms via ether cleavage of triclosan. In experiments with excess free chlorine, 2,4,6-trichlorophenol was formed via electrophilic substitution of 2,4-dichlorophenol. Chloroform formation was observed when an excess of free chlorine was present. A Hammett-type linear free-energy relationship (LFER) using Brown-Okamoto parameters (sigma+) was established to correlate the reactivity of HOCI and the phenolate forms of triclosan and other chlorophenols (log kArO- = -(10.7 +/- 2.2)Sigmasigma(+)o,m,p + 4.43). This LFER was used to obtain estimates of rate coefficients describing the reactivity of the intermediates 5,6-dichloro-2-(2,4-dichlorophenoy)phenol (kArO- approximately equal to 6 x 10(2)), 4,5-dichloro-2-(2,4-dichlorophenoxy)phenol (kArO- approximately equal to 3 x 10(2)), and 4,5,6-trichloro-(2,4-dichlorophenoxy)phenol (kArO- approximately equal to 4 x 10(1)).

Anti-Infective Agents, Local↗
PubMed2004

Adsorption of aromatic compounds from water by treated carbon materials.

Carbon materials with different textural and surface chemistry properties have been studied to analyze their behavior in removing aromatic compounds (phenol, o-chlorophenol, p-nitrophenol, aniline, and phenol compound mixtures) from water. A mesoporous high surface area graphite and a microporous activated carbon with (HSAGox and ACox) and without (HSAGT and ACT) oxygen surface groups, were used as adsorbents. Apparent surface areas, surface oxygen groups, and zero points of charge have been determined. The adsorption behavior of single compounds on ACT depends on the relation between the molecular and the pore sizes. The aniline, the nitrophenol, and the chlorophenol interact with the oxygen surface groups of oxidized graphite, while there is no evidence of any type of interaction of the phenol with these groups. The adsorption of the organic compound mixtures on the thermally treated samples is determined by the acid-base character of the adsorbate-adsorbent, whereas on the oxidized carbons, the controlling forces are the specific interactions between organic molecules and the oxygenated groups. Selectivity coefficients for the different mixtures are presented over the entire range of adsorption.

Adsorption↗
PubMed2006

Cobalt-mediated activation of peroxymonosulfate and sulfate radical attack on phenolic compounds. implications of chloride ions.

The sulfate radical pathway of the room-temperature degradation of two phenolic compounds in water is reported in this study. The sulfate radicals were produced by the cobalt-mediated decomposition of peroxymonosulfate (Oxone) in an aqueous homogeneous system. The major intermediates formed from the transformation of 2,4-dichlorophenol were 2,4,6-trichlorophenol, 2,3,5,6-tetrachloro-1,4-benzenediol, 1,1,3,3-tetrachloroacetone, pentachloroacetone, and carbon tetrachloride. Those resulting from the transformation of phenol in the presence of chloride ion were 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 1,1,3,3-tetrachloroacetone, and pentachloroacetone. In the absence of chloride ion, phenol transformed into 2,5-cyclohexadiene-1,4-dione (quinone), 1,2-benzenediol (catechol), and 1,4-benzenediol (hydroquinone). Several parameters were varied, and their impact on the transformation of the organic compounds is also discussed. The parameters varied were the initial concentration of the organic substrate, the dose of Oxone used, the cobalt counteranion, and in particular the impact of chloride ions and the quenching agent utilized for terminating the reaction. This is one of the very few studies dealing with intermediates formed via sulfate radical attack on phenolic compounds. It is also the first studythat explores the sulfate radical mechanism of oxidation, when sulfate radicals are generated via the Co/Oxone reagent. Furthermore, it provides strong evidence on the interaction of chloride ions with sulfate radicals leading to halogenation of organics in water.

Chlorides↗
PubMed2003

Ochratoxin a forms a carbon-bonded c8-deoxyguanosine nucleoside adduct: implications for c8 reactivity by a phenolic radical.

The ability of the carcinogenic fungal toxin Ochratoxin A (OTA, 1) to react with deoxyguanosine (dG) has been assessed using electrospray mass spectrometry and NMR. Photoexcitation of OTA (100 muM) in the presence of 50 mol equiv of dG led to the isolation and identification of the C8-deoxyguanosine nucleoside adduct 4. Importantly, the same adduct was formed upon oxidative activation of OTA using horseradish peroxidase (HRP)/H2O2 or the transition metals Fe(II) and Cu(II), as evidenced by mass spectrometry. Because the mutagenicity and subsequent carcinogenicity of OTA are believed to stem from oxidative DNA damage (strand scission and oxidative base products) and formation of guanine-specific DNA adducts, the adduct 4 confirms the ability of OTA to react covalently with dG and has important implications for the mechanism of action of OTA and other chlorophenolic toxins that undergo oxidation to yield phenoxyl radicals. The C8 position of dG is susceptible to radical attack, as was amply proven through formation of the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine. The adduct 4 is the first structurally characterized nucleoside adduct of a chlorophenolic toxin, and its formation has important implications for the mutagenicity of phenolic xenobiotics.

Carcinogens↗
PubMed1999

Nitrosation of phenolic compounds: inhibition and enhancement.

The nitrosation of phenol, m-, o-, and p-cresol, 2,3-, 3,5-, and 2, 6-dimethylphenol, 3,5-di-tert-butylphenol, 2,4,6-trimethylphenol, o-chlorophenol, and o-bromophenol was studied. Kinetic monitoring of the reactions was accomplished by spectrophotometric analysis of the products at 345 nm. At pH > 3, the dominant reaction was C-nitrosation through a mechanism that appears to consist of an attack on the nitrosatable substrate by NO(+)/NO(2)H(2)(+), followed by a slow proton transfer. The finding of an isokinetic relationship supports the idea that the same mechanism operates throughout the series. The observed sequence of nitrosatable substrate reactivities is explained by (i) the preferred para-orientation of the hydroxyl group for the electrophilic attack of nitrosating agents, (ii) steric hindrance of alkyl substituents, which reduces or prevents attack by nitrosating agents, and (iii) the hyperconjugative effect of the methyl substituent, which causes electronic charge to flow into the aromatic nucleus, as well as the opposite electronic withdrawing effect induced by halogen substituents. The results show that potential nitrosation of widespread environmental species such as chlorophenols is negligible, but more attention should be paid to polyphenols with strongly nucleophilic carbon atoms.

Cresols↗
PubMed2004

Electrophilic amination of 4-fluorophenol with diazenes: a complete removal of the fluorine atom.

The electrophilic amination of 2-fluorophenol, 4-fluorophenol, and 2-chlorophenol was observed to occur as a result of their treatment with diazenes 1-4 under mild reaction conditions in the presence of ZrCl(4). The products originating from the 2-fluorophenol or 2-chlorophenol can be considered as "normal" products of amination. On the other hand, the 2-chloro-4-amino-substituted phenols obtained from the 4-fluorophenol seem to be formed in a process that involves an ipso amination, the complete removal of the fluorine atom, and the introduction of the chlorine atom.

Journal Article↗
PubMed2005

Molecular structures and properties of the complete series of bromophenols: Density functional theory calculations.

The complete series of 19 bromophenols have been studied by density functional theory (DFT) calculations at the B3LYP/6-311G++(d,p) level. The molecular structures and properties of bromophenols are strongly influenced by intramolecular hydrogen bonding of ortho-bromine, steric and inductive effects of substituted bromine, and other intramolecular electrostatic interactions. Systematic trends in several structural parameters and molecular properties of bromophenols have been found with the increasing number of bromine substitutions, including increase in O-H bond length, decrease in C-O bond length, red shift in O-H stretching frequency, and blue shift in O-H torsional frequency. Correlations among several key molecular parameters as well as those with available aqueous pKa values are examined. Comparisons with chlorophenols have indicated that the inductive effect of substituted bromine appears larger and bromophenols are slightly stronger acids than chlorophenols.

Bromine↗
PubMed2006

Effects of polyoxometalate and fluoride on adsorption and photocatalytic degradation of organic dye X3B on TiO2: the difference in the production of reactive species.

It has been reported that addition of polyoxometalates (POM) or fluoride anions into the TiO(2) dispersions can significantly enhance the photocatalytic degradation (PCD) of weakly adsorptive organic pollutants in water such as chlorophenol. In this work, however, contradictory effects of POM and fluoride were observed on the PCD of highly adsorptive substrate X3B, an anionic organic dye, under similar conditions. The total rate of X3B PCD, determined by total loss of X3B both in solution and on the catalyst surface, was increased in the presence of fluoride, but the rate was decreased in the presence of POM. In both cases, the dark adsorption of X3B on TiO(2) was greatly decreased, ascribed to competitive adsorption of POM or fluoride that reduces the positive charges on the catalyst surface. The spectral analysis and the kinetic study using tert-butyl alcohol as hydroxyl radical scavenger revealed that the PCD of X3B on naked TiO(2) was predominately initiated by direct hole transfer, whereas addition of POM or fluoride into the TiO(2) dispersions enhanced the degradation of X3B via hydroxyl radical pathway. It is proposed that the surface occupation of POM on TiO(2) accelerates the production of surface-bound hydroxyl radicals, due to enhanced charge separation, whereas the fluoride replacement of surface hydroxyl groups of TiO(2) increases the production of free hydroxyl radicals in solution, due to enhanced hole availability for water oxidation. Assume that the relative reactivity among various active follows the order of free hydroxyl radicals > subsurface holes > surface-bound hydroxyl radicals, the proposal could account for the observed effects of POM and fluoride on the PCD of both weakly and highly adsorptive organic substrates over TiO(2) such as chlorophenol and X3B.

Journal Article↗
PubMed2005

Separating surface and solvent effects and the notion of critical adsorption energy in the adsorption of phenolic compounds by activated carbons.

A modified form of the Freundlich equation in which the solute equilibrium concentration is normalized with respect to the solute solubility is analyzed and applied to adsorption isotherms of phenol, 4-nitrophenol, 4-chlorophenol, and 2-chlorophenol at different values of pH on commercial activated carbon before and after oxidation. The analysis confirms the importance of normalizing the solute equilibrium concentration when analyzing the adsorption isotherms, and it is suggested that a parameter, K(F10), obtained by taking 10% solubility as the reference point when applying the Freundlich equation, is probably the best comparative estimate of the relative adsorption capacity of the carbon for different phenolic compounds. In combination with the Freundlich exponent, n(F), estimates of the adsorption capacity at any other reference point can then be obtained. Analysis of the experimental results also indicates a need to distinguish between two regimes of adsorption, characterized by an adsorption energy, E(ads), greater than or less than a critical value, E(ca). When E(ads) > E(ca), the shape of the adsorption isotherm is determined by solute-solid interactions. On the other hand, when E(ads) < E(ca), solute-solution interactions become more important.

Adsorption↗
PubMed1999

Preliminary characterization of four 2-chlorobenzoate-degrading anaerobic bacterial consortia.

Dechlorination was the initial step of 2CB biodegradation in four 2-chlorobenzoate-degrading methanogenic consortia. Selected characteristics of ortho reductive dehalogenation were examined in consortia developed from the highest actively dechlorinating dilutions of the original 2CB consortia, designated consortia M34(-9), P20(-9), P21(-9) and M50(-7). In addition to 2-chlorobenzoate, all four dilution consortia dehalogenated 4 of 32 additional halogenated aromatic substrates tested, including 2-bromobenzoate; 2,6-dichlorobenzoate; 2,4-dichlorobenzoate; and 2-chloro-5-hydroxybenzoate. Dehalogenation occurred exclusively at the ortho position. Both ortho chlorines were removed from 2,6-dichlorobenzoate. Benzoate was detected from 2-bromobenzoate and 2,6-dichlorobenzoate. 4-Chlorobenzoate and 3-hydroxybenzoate were formed from 2,4-dichlorobenzoate and 2-chloro-5-hydroxybenzoate, respectively. Only benzoate was further degraded. Slightly altering the structure of the parent "benzoate molecule" resulted in observing reductive biotransformations other than dehalogenation. 2-Chlorobenzaldehyde was reduced to 2-chlorobenzyl alcohol by all four consortia. 2-chloroanisole was O-demethoxylated by three of the four consortia forming 2-chlorophenol. GC-MS analysis indicated reduction of the double bond in the propenoic side chain of 2-chlorocinnamate forming 2-chlorohydrocinnamate. None of the reduction products was dechlorinated. The following were not dehalogenated: 3- and 4-bromobenzoate; 3- and 4-chlorobenzoate; 2-, 3-, and 4-fluorobenzoate; 2-, 3-, and 4-iodobenzoate; 2-, 3-, and 4-chlorophenol; 2-chloroaniline; 2-chloro-5-methylbenzoate; 2,3-dichlorobenzoate; 2,5-dichlorobenzoate; 2,4,5-trichlorophenoxyacetic acid; and 2,4-dichlorophenoxyacetic acid. Consortia M34(-9), P20(-9), P21(-9), and M50(-7) dechlorinated 2-chlorobenzoate at < or = 4 mm. Dechlorination rates were highest for consortia P20(-9) followed by those of M50(-7) with rates declining above 2 and 3 mm 2CB, respectively. The major physiological types of microorganisms in consortia M34(-9), P20(-9), P21(-9), and M50(-7) were sulfate-reducing and hydrogen-utilizing anaerobes.

Bacteria, Anaerobic↗
PubMed1999

A comparison of the ability of forest and agricultural soils to mineralize chlorinated aromatic compounds.

Soils were sampled from two agricultural fields, two relatively pristine forests, and one suburban forest in Ontario, Canada. The ability of these soils to mineralize 2,4-dichlorophenoxyacetate, 3-chlorobenzoate, 4-chlorophenol, 2,4-dichlorophenol, pentachlorophenol, and atrazine was determined using 14C-labeled substrates. Direct pre-exposure was necessary before atrazine mineralization could be detected; however, it was not necessary for degradation of any of the other chemicals. 2,4-dichlorophenoxyacetate and pentachlorophenol mineralization was much higher in the agricultural soils relative to the pristine forest soils, but 3-chlorobenzoate and 2,4-dichlorophenol mineralization rates showed the opposite trend. Mineralization of 4-chlorophenol was about equivalent in all soils. Suburban forests soils were indistinguishable from agricultural soils with respect to their degradation of 2,4-dichlorophenoxyacetate and chlorobenzoate. Additionally, they were better able than any of the soils to withstand the toxic effects of pentachlorophenol. Pentachlorophenol mineralization was highly variable in the pristine forest soils, ranging from about 6 to 50%. Abiotic factors such as pH, soil type, and organic and moisture content did not account for these significant site differences. The selective forces responsible for these differences, and the possible differences in microbial populations are discussed.

Agriculture↗
PubMed2003

Sulphate-reducing bacteria, palladium and the reductive dehalogenation of chlorinated aromatic compounds.

The surfaces of cells of Desulfovibrio desulfuricans, Desulfovibrio vulgaris and a new strain, Desulfovibrio sp. 'Oz-7' were used to manufacture a novel bioinorganic catalyst via the reduction of Pd(II) to Pd(0) at the cell surface using hydrogen as the electron donor. The ability of the palladium coated (palladised) cells to reductively dehalogenate chlorophenol and polychlorinated biphenyl species was demonstrated. Dried, palladised cells of D. desulfuricans, D. vulgaris and Desulfovibrio sp. 'Oz-7' were more effective bioinorganic catalysts than Pd(II) reduced chemically under H2 or commercially available finely divided Pd(0). Differences were observed in the catalytic activity of the preparations when compared with each other. Negligible chloride release occurred from chlorophenol and polychlorinated biphenyls using biomass alone.

Bacteria↗
PubMed2002

Adsorption and detection of some phenolic compounds by rice husk ash of Kenyan origin.

Rice husk ash (RHA) obtained from a rice mill in Kenya has been used as an inexpensive and effective adsorbent (and reagent) for the removal (and detection) of some phenolic compounds in water. The abundantly available rice mill waste was used in dual laboratory-scale batch experiments to evaluate its potential in: (i) the removal of phenol, 1,3-dihydroxybenzene (resorcinol) and 2-chlorophenol from water; and (ii) the detection of 1,2-dihydroxybenzene (pyrocatechol) and 1,2,3-trihydroxybenzene (pyrogallol) present in an aqueous medium. The studies were conducted using synthetic water with different initial concentrations of the phenolic compounds. The effects of different operating conditions (such as contact time, concentration of the phenolic compounds, adsorbent quantity, temperature, and pH) were assessed by evaluating the phenolic compound removal efficiency as well as the extent of their color formation reactions (where applicable). RHA exhibits reasonable adsorption capacity for the phenolic compounds and follows both Langmuir and Freundlich isotherm models. Adsorption capacities of 1.53 x 10(-4), 8.07 x 10(-5), and 1.63 x 10(-6) mol g(-1) were determined for phenol, resorcinol and 2-chlorophenol, respectively. Nearly 100% adsorption of the phenolic compounds was possible and this depended on the weight of RHA employed. For the detection experiments, pyrocatechol and pyrogallol present in water formed coloured complexes with RHA, with the rate of colour formation increasing with temperature, weight of RHA, concentration of the phenolic compounds and sonication. This study has proven that RHA is a useful agricultural waste product for the removal and detection of some phenolic compounds.

Adsorption↗
PubMed2003

Comparison of the effects of UV, H2O2/UV and gamma-irradiation processes on frozen and liquid water solutions of monochlorophenols.

The effects of UV irradiation, both in the presence and absence of hydrogen peroxide, as well as of gamma irradiation on 2- and 4-chlorophenol in a solid water ice matrix have been studied and compared to those effects known to occur in aqueous solutions. While UV photolysis (>280 nm) of monochlorophenols leads to efficient coupling reactions in ice and photosolvolysis products in liquid water; hydroxylation to chlorobenzenediols is the main pathway in the presence of H2O2 in both phases. The results show that the solute molecules accumulate in a layer surrounding the ice crystal walls during the freezing process, where they then react. The radiation chemistry of chlorophenol ice samples involves preferential coupling reactions at -78 degrees C rather than reactions with the OH radicals produced by cleavage of water molecules under the conditions employed (1 kGy h(-1)). The apparent similarities between the chemistry in the UV/H2O2-treated liquid and solid, and y-irradiated liquid and solid samples are discussed. It is suggested that the reactions of OH radicals within polycrystalline ice or snow are important natural processes that should be considered in environmental, ice-core or astrophysical research.

Journal Article↗
PubMed2002

Successive rapid reductive dehalogenation and mineralization of pentachlorophenol by the indigenous microflora of farmyard manure compost.

AIMS: To determine whether composting with animal manure can be used to effectively remediate soil from a pentachlorophenol (PCP)-contaminated site, and to establish the fate of the degraded xenobiotic. METHODS AND RESULTS: Contaminated soil from a sawmill site was mixed with farm animal manure and composted in a 0.5 m3 silo under fully aerobic conditions. The disappearance and fate of PCP was monitored by gas chromatography (GC-ECD) and extensive mineralization confirmed in experiments with 14C-radiolabelled PCP. The disappearance of PCP was rapid and virtually complete within 6 days, prior to the onset of thermophilic conditions. Dechlorination of the PCP was found to be both reductive and sequential. CONCLUSIONS: PCP removal from contaminated soil by aerobic composting with animal manure is efficient and proceeds via reductive dechlorination to virtually complete mineralization. This contrasts with other chlorophenol composting regimes in which mineralization is achieved but dechlorination intermediates do not accumulate to detectable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study demonstrate that anaerobic reductive dechlorination can proceed in an aerobic composting environment and contribute to efficient pentachlorophenol removal. Farmyard manure composts may represent a rapid, low-cost, low-technology option for treatment of chlorophenol-contaminated soils.

Agriculture↗
PubMed2001

Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450(cam)).

Polychlorinated benzenes are recalcitrant environmental pollutants primarily because they are resistant to attack by dioxygenases commonly used by micro-organisms for the biodegradation of aromatic compounds. We have investigated the oxidation of polychlorinated benzenes by mutants of the haem mono-oxygenase CYP101 (cytochrome P450(cam)) from Pseudomonas putida with the aim of generating novel systems for their biodegradation. Wild-type CYP101 had low activity for the oxidation of dichlorobenzenes and trichlorobenzenes to the chlorophenols, but no products were detected for the heavily chlorinated benzenes. Increasing the active-site hydrophobicity with the Y96F mutation increased the activity up to 100-fold, and both pentachlorobenzene and hexachlorobenzene were oxidized slowly to pentachlorophenol. Decreasing the space available at the top of the active site with the F87W mutation to force the substrate to be bound closer to the haem resulted in a further 10-fold increase in activity with most substrates. Introducing the F98W mutation, also at the top of the active site, decreased the NADH-turnover rates but increased the coupling efficiencies, and > 90% coupling was observed for 1,3-dichlorobenzene and 1,3,5-trichlorobenzene with the F87W--Y96F--F98W mutant. The V247L mutation generally increased the NADH-turnover rates, and the F87W--Y96F--V247L mutant showed reasonably fast NADH turnover (229 min(-1)) with the highly insoluble pentachlorobenzene without the need for surfactants or organic cosolvents. As all chlorophenols are degraded by micro-organisms, novel biodegradation systems could be constructed in which CYP101 mutants convert the inert polychlorinated benzenes to the phenols, which are then readily degraded by natural pathways.

Benzene Derivatives↗