Laccase-mediated detoxification of phenolic compounds

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Oxidative coupling of aromatic pesticide intermediates by a fungal phenol oxidase.

The soil fungus Rhizoctonia praticola produced an enzyme that accumulated in the growth medium and caused the polymerization of phenolic and naphtholic intermediates of various pesticides. The dialyzed crude enzyme was purified by ion-exhange column chromatography with diethylaminoethyl-cellulose, followed by gel filtration with Sephadex G-200. The enzyme, a phenol oxidase, was capable of polymerizing 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, and 4-bromo-2-chlorophenol. 1-Naphthol, 2-naphthol, and some of their derivatives formed oligomers or polymers when incubated with the enzyme, but 4-nitrophenol and 2,4-dinitriphenol were not oxidized. Chlorinated and brominated anilines, which are derivatives of herbicides, were not altered by the phenol oxidase from R. praticola, but 4-methoxyaniline was transformed by the enzyme to 2-amino-5-p-anisidinobenzoquinone-di-p-methoxyphenylimine. The formation of polymeric products was determined by mass spectrometric analysis.     

Asymmetric diphenol formation by a fungal laccase.

A laccase isolated from the fungus Rhizoctonia praticola catalyzed the cross-coupling of two differently halogenated phenols. When 2,4-dichlorophenol and 4-bromo-2-chlorophenol were incubated together with the enzyme, three dimers were formed and isolated by thin-layer chromatography. The molecular weights of these compounds were determined by mass spectrometry as 322, 410, and 366, which correspond with the respective dimers of each of the phenols and with a hybrid formed from both, tentatively assigned the structure 3,3',5'-trichloro-5-bromo-2,2'-diphenol. Gas chromatography-mass spectrometry analysis of these products and of their methylated derivatives lent support to these structural assignments.     

Asymmetric diphenol formation by a fungal laccase.

A laccase isolated from the fungus Rhizoctonia praticola catalyzed the cross-coupling of two differently halogenated phenols. When 2,4-dichlorophenol and 4-bromo-2-chlorophenol were incubated together with the enzyme, three dimers were formed and isolated by thin-layer chromatography. The molecular weights of these compounds were determined by mass spectrometry as 322, 410, and 366, which correspond with the respective dimers of each of the phenols and with a hybrid formed from both, tentatively assigned the structure 3,3',5'-trichloro-5-bromo-2,2'-diphenol. Gas chromatography-mass spectrometry analysis of these products and of their methylated derivatives lent support to these structural assignments.     

Enhanced removal of three phenols by laccase polymerization with MF/UF membranes

Guaiacol, catechol, m-cresol are common phenolic compounds presented in various industrial effluents but difficult to be removed by conventional wastewater treatment schemes. To elucidate mechanisms of enhanced membrane removal by laccase polymerization, different MF and UF membranes were employed in a cross-flow module for phenol concentration of 5mM. With 2.98 IU/l of laccase applied at room temperature, guaiacol, catechol and m-cresol were polymerized to products of averaged molecular weight of 9600, 8350 and 5400 Da (Dalton), respectively. Methoxy and hydroxyl-substituted phenols (guaiacol and catechol) were polymerized better than methyl-substituted phenol (m-cresol) due to more stable free-radical containing intermediate structure induced by oxygen-containing methoxy and hydroxyl functional groups. Removal efficiencies for the un-reacted phenols were dependent on the molecular sizes (length and width), but were dependent on the molecular weight for the polymerized phenolic compounds. Flux was declined initially but reached steady state after 180 min of filtration, indicating these MF/UF membranes can be used for removal of these polymerized phenols without significant fouling. In addition, pretreatments by the inactivated laccase only caused further flux reduction without additional removal of phenols.     

Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.     

Degradation of mixtures of phenolic compounds by <Emphasis Type="Italic">Arthrobacter chlorophenolicus</Emphasis> A6

In this study the chlorophenol-degrading actinobacterium, Arthrobacter chlorophenolicus A6, was tested for its ability to grow on mixtures of phenolic compounds. During the experiments depletion of the compounds was monitored, as were cell growth and activity. Activity assays were based on bioluminescence output from a luciferase-tagged strain. When the cells were grown on a mixture of 4-chlorophenol, 4-nitrophenol and phenol, 4-chlorophenol degradation apparently was delayed until 4-nitrophenol was almost completely depleted. Phenol was degraded more slowly than the other compounds and not until 4-nitrophenol and 4-chlorophenol were depleted, despite this being the least toxic compound of the three. A similar order of degradation was observed in non-sterile soil slurries inoculated with A. chlorophenolicus. The kinetics of degradation of the substituted phenols suggest that the preferential order of their depletion could be due to their respective pKa values and that the dissociated phenolate ions are the substrates. A mutant strain (T99), with a disrupted hydroxyquinol dioxygenase gene in the previously described 4-chlorophenol degradation gene cluster, was also studied for its ability to grow on the different phenols. The mutant strain was able to grow on phenol, but not on either of the substituted phenols, suggesting a different catabolic pathway for the degradation of phenol by this microorganism.     

Enhanced transformation of malachite green by laccase of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the presence of natural phenolic compounds

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.     

Development of a Saccharomyces cerevisiae Strain with Enhanced Resistance to Phenolic Fermentation Inhibitors in Lignocellulose Hydrolysates by Heterologous Expression of Laccase

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.     

Fungal bioremediation of phenolic wastewaters in an airlift reactor

Of the various types of industry-generated effluents, those containing organic pollutants such as phenols are generally difficult to remediate. There is a need to develop new technologies that emphasize the destruction of these pollutants rather than their disposal. In this work the white rot fungus, Trametes pubescens, was demonstrated to be an effective bioremediation agent for the treatment of phenolic wastewaters. An airlift loop reactor was optimized, in terms of volumetric oxygen transfer rate (K(L)a = 0.45 s(-1)), to provide an environment suited to rapid growth of T.pubescens (mu = 0.25 day(-1)) and a particularly efficient growth yield on glucose of 0.87 g biomass.g glucose(-1). The phenolic effluent was shown to be a paramorphogen, influencing fungal pellet morphology in the reactor, as well as increasing laccase enzyme activity by a factor of 5 over the control, to a maximum of 11.8 U.mL(-1). This increased activity was aided by the feeding of nonrepressing amounts (0.5 g.L(-1)) of glucose to the reactor culture. To our knowledge the degradation results represent the highest rate of removal (0.033 g phenol.g biomass(-1).day(-1)) of phenolic compounds from water reported for white rot fungi.     

First evidence of catalytic mediation by phenolic compounds in the laccase-induced oxidation of lignin models.

The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance-stabilized phenoxy radical, performs as a mediator in the laccase-catalyzed oxidation of a nonphenolic substrate (4-methoxybenzyl alcohol) and also of a hindered phenol (2,4,6-tri-tert-butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes.     

Response of sweet pepper to phenols accumulated in greenhouse substrate

Prolonged cucumber cultivation in the same substrates leads to accumulation of phytotoxic phenolic compounds. Introduction of sweet pepper as an aftercrop eliminates substrate phytotoxicity. The aim of the study was to examine whether in sweet pepper detoxication of substrate phenols occurs by means of the glucosylation process. The examined materials were substrates differing in phytotoxicity level, and sweet pepper plants grown on these substrates. Substrate phytotoxicity was obtained by means of either repeated cucumber cultivation or by phenolic acid addition. During the vegetative growth phase of sweet pepper, the phytotoxicity and phenolic compound levels in the substrate, and the glucosylated phenol contents in above-ground plant parts were determined. Results showed that sweet pepper responds to an increased presence of phenols in the substrate by an intensified glucosylation.     

Enzymatic modification of kraft lignin through oxidative coupling with water-soluble phenols

The aromatic polymer lignin can be modified through promotion of oxidative coupling between phenolic groups on lignin and various phenols. The reaction is initiated by an oxidation of both components, e.g., by using the oxidoreductases laccase or peroxidase. Coupling between phenolic monomers and lignin has previously been studied by the use of radio-labeled phenols. In this study, incorporation of water-soluble phenols into kraft lignin, using laccase as catalyst, was investigated. Several phenols with carboxylic or sulfonic acid groups were used as markers for the incorporation. The modified lignin was isolated and the amount of phenol incorporated was characterized by means of titration, quantitative 1H-NMR, and quantitative 31P-NMR after modification with 2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphospholane. Only a few of the phenols studied were found to be incorporated into lignin. When the phenol guaiacol sulfonate was incorporated into kraft lignin, the lignin became water-soluble at pH 2.4 and a low ionic strength due to the introduction of sulfonic acid groups. The content of sulfonic acid groups in the product was 0.5-0.6 mmol/g lignin. A lower amount of 4-hydroxyphenylacetic acid was incorporated under similar conditions.     

Enhanced activity by poly(ethylene glycol) modification of Coriolopsis gallica laccase

We are studying the enzymatic modification of polycyclic aromatic hydrocarbons (PAHs) by the laccase from Coriolopsis gallica UAMH 8260. The enzyme was produced during growth in a stirred tank reactor to 15 units ml⁻¹, among the highest levels described for a wild-type fungus; the enzyme was the major protein produced under these conditions. After purification, it exhibited characteristics typical of a white rot fungal laccase. Fifteen azo and phenolic compounds at 1 mM concentration were tested as mediators in the laccase oxidation of anthracene. Higher anthracene oxidation was obtained with the mediator combination of ABTS and HBT, showing a correlation between the oxidation rate and the mediator concentration. Reactions with substituted phenols and anilines, conventional laccase substrates, and PAHs were compared using the native laccase and enzyme preparations chemically modified with 5000 MW-poly(ethylene glycol). Chemically modified laccase oxidized a similar range of substituted phenols as the native enzyme but with a higher catalytic efficiency. The k _cat increase by the chemical modification may be as great as 1300 times for syringaldazine oxidation. No effect was found of chemical modification on mediated PAH oxidation. Both unmodified and PEG-modified laccases increased PAH oxidation up to 1000 times in the presence of radical mediators. Thus, a change of the protein surface improves the mediator oxidation efficiency, but does not affect non-enzymatic PAH oxidation by oxidized mediators. Received 10 December 2001/ Accepted in revised form 20 July 2002     

Phenols removal in musts: Strategy for wine stabilization by laccase

The potential of laccase from Trametes versicolor for phenolic removal in must for wine stabilization was evaluated through a combination of an analytical methodology (capillary zone electrophoresis) and kinetics of phenols removal as the total antioxidant potential variation. Total phenolic content, total antioxidant potential and polyphenols were monitored from 0 to 3 h of must treatment. The results indicated that the treatment of a red must with laccase affect mainly the phenolic compounds responsible for the must antioxidant properties. The treatment of white musts with laccase showed higher reduction in total phenol than in the total antioxidant potential. Phenol degradation by laccase was very fast for catechins, and slowly for stilbenes (cis- and trans-resveratrol) and derivatives of cinnamic (ferulic and caffeic) and benzoic (syringic, vanillic, and gallic) acids. It is possible to conclude in this case that the use of laccase in white wines is perfectly feasible. This would allow softer and ecologically correct treatments, which would diminish the cost of processing and avoid deterioration of wines for long storage times.     

Characterization of an enzyme from Rhizoctonia praticola which polymerizes phenolic compounds.

An extracellular phenol oxidase from the fungus Rhizoctonia praticola which polymerizes various xenobiotic phenols was isolated and characterized. The enzyme was purified by DEAE-cellulose and Sephadex G-200 chromatography followed by preparative polyacrylamide gel electrophoresis. Atomic absorption and EPR spectroscopy indicated the presence of copper, and SDS gel electrophoresis revealed a molecular weight of 78,000. With 2,6-dimethoxyphenol as substrate, the enzyme showed a pH optimum of 6.7--6.9, and a temperature optimum of 40 degrees C. According to these and additional characteristics it appears that the enzyme belongs to the class of laccases.     

Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent

There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.     

Determination of phenolic compounds by a polyphenol oxidase amperometric biosensor and artificial neural network analysis

The determination of phenolic compounds is significant given its toxicity, even at very low concentration levels. Amperometric determination of phenols is a simple technique available. Direct oxidation of phenols can be used, but another possibility is the use of polyphenol oxidase (tyrosinase) enzyme biosensors that oxidises the phenolic compounds into their corresponding quinones. Reduction of the resulting quinones accomplishes the amplification of the amperometric signal, as long as the result of the reduction process is the corresponding cathecol, this being able to be oxidised again by the polyphenol oxidase immobilized on the surface of the biosensor. In this communication, simultaneous determination of different phenols was carried out combining biosensor measurements with chemometric tools, in what is known as electronic tongue. The departure information used was the overlapped reduction voltammogram generated with the amperometric biosensor based on polyphenol oxidase. Artificial Neural Networks (ANN) were used for extraction and quantification of each compound. Phenol, cathecol and m-cresol formed the three-analyte study case resolved in this work. Good prediction ability was attained, and so, the separate quantification of these three phenols was accomplished.     

Screening of fungal isolates and properties of Ganoderma applanatum intended for olive mill wastewater decolourization and dephenolization

AIMS: To investigate different autochthonous isolates of wood-rotting fungi for the removal of both colour and phenolic compounds from olive mill wastewaters (OMW). METHODS AND RESULTS: The isolates Bjerkandera adusta Ba-100, Fomes fomentarius Ff-106, Ganoderma applanatum Ga-20, Irpex lacteus Il-3, Trametes versicolor Tv-101 and Tv-103 were preliminarily screened for their OMW-decolourizing potential on potato dextrose agar supplemented with different OMW concentrations. A further screening of batch cultures under different agitation speeds, to test the effect of shear stress, resulted in the selection of isolate G. applanatum Ga-20. Batch cultures grown in OMW-based medium exhibited strong laccase induction and significant decrease in the values of phenols, colour and chemical oxygen demand. Concomitant onset of laccase activity and colour removal was observed, and apart from laccase, neither lignin peroxidase nor manganese-dependent peroxidase activities were detected. Moreover, the depletion of aromatic compounds with high and low apparent molecular mass was observed by chromatographic analysis. CONCLUSIONS: Isolate G. applanatum Ga-20 exhibited interesting properties for its use in bioremediation of OMW, namely high removal of recalcitrant phenolic compounds and strong colour abatement. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the white-rot fungus G. applanatum proves to be effective for the decolourization and dephenolization of OMW.