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.     

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.     

Allelopathic Interactions Involving Phenolic Acids

A major concern regarding allelopathic interactions involving phenolic acids in no-till systems pertains to the fact that concentrations of individual phenolic acids recoverable from field soils are well below levels required for inhibition of germination and seedling growth in laboratory bioassays. Field soils contain a variety of phenolic acids as well as other toxic and nontoxic organic compounds that are available to interact with seeds and roots; whereas in laboratory bioassays, with few exceptions, single phenolic acids have been tested. Studies of mixtures of phenolic acids and other toxic (e.g., methionine) and nontoxic (e.g., glucose) organic compounds in laboratory bioassays indicate that the action of a single phenolic acid is not representative of the actions of such mixtures. Specifically, as the number of phenolic acids added to soil increased, concentrations of the individual phenolic acids required to bring about a growth inhibition declined. The addition of other organic compounds (e.g., glucose, methionine) to the soil also reduced the concentration of a phenolic acid (e.g., p-coumaric acid) required for growth inhibition. These results support the hypothesis that in the field mixtures of phenolic acids and other organic compounds can cause inhibitory effects even though the concentrations of individual compounds are well below their inhibitory levels.     

Redox‐Active Phenolic Compounds Mediate the Cytotoxic and Antioxidant Effects of Carpodesmia tamariscifolia (=Cystoseira tamariscifolia)

Carpodesmia tamariscifolia is a brown alga rich in (poly)phenols with important cytotoxic and antioxidant effects. However, the relationship between its chemical composition and its effects is unknown. The aim of this study is to identify the potential compounds and mechanisms responsible for its main effects. The alga was extracted consecutively with hexane, dichloromethane and methanol and further fractionated using Sephadex LH‐20 and silica gel columns when appropriate. The fractions were subjected to thin‐layer chromatography and liquid chromatography‐mass spectrometry analysis and evaluated for their total phenolic content (Folin‐Ciocalteu assay), radical scavenging activity (DPPH assay), cytotoxic activity (MTT assay on the SH‐SY5Y cell line), and ability to generate H₂O₂ (Amplex Red assay). Chromatographic and phenolic analyses of the fractions indicate that abundant redox‐active phenols are present in all the fractions and that a high amount of prenylated hydroquinone derivatives is present in the apolar ones. In the hexane and dichloromethane fractions, the cytotoxic and antioxidant activities are closely related to their phenolic content, whereas in the methanol fractions, the cytotoxicity is negatively related to the phenolic content and the antioxidant activity is positively related to it. In the same tests, hydroquinone behaves as both strong cytotoxic and antioxidant agent. H₂O₂ assay shows that C. tamariscifolia fractions and hydroquinone can autoxidize and generate H₂O₂. Our results suggest that redox‐active phenols produce the pharmacological effects described for C. tamariscifolia and that the hydroquinone moiety of prenylated hydroquinone derivatives is responsible for both cytotoxic (through a pro‐oxidant mechanism secondary to its autoxidation) and antioxidant effects of the apolar fractions.     

Flavin-containing monooxygenases from <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> responsible for fungal metabolism of phenolic compounds

We investigated the cellular responses of the white-rot basidiomycete Phanerochaete chrysosporium against vanillin. Based upon a proteomic survey, it was demonstrated that two flavin-containing monooxygenases (PcFMO1 and PcFMO2) are translationally up-regulated in response to exogenous addition of vanillin. To elucidate their catalytic functions, we cloned cDNAs and heterologously expressed them in Escherichia coli. The recombinant PcFMO1 showed catalytic activities against monocyclic phenols such as phenol, hydroquinone, and 4-chlorophenol. In addition, the product from hydroquinone was identified as 1,2,4-trihydroxybenzene, an important intermediate in a metabolic pathway of aromatic compounds in which the aromatic ring of 1,2,4-trihydroxybenzene can be further cleaved by fungal dioxygenases for mineralization. Thus, the ortho-cleavage pathway of phenolic compounds would presumably be associated with PcFMO1.     

The Influence of Some Phenolic Compounds on Nodulation in Pigeonpea (Cajanus cajan(L.) Millsp.)

Studies on exogenous application of phenolic compoundsviz: p-hydroxybenzoic acid, resorcinol and chlorogenic acid each with concentration of 10^-4 M are done on the legume (Cajanus cajan (L.)Millsp.) AL-15. The effect of applied phenolic compounds as well as of structural differences in phenols indicate a marked influence of phenolic compounds in regulating growth processes in plants. Fresh and dry mass of various plant parts increased after foliar spray with phenols resulting in an improved harvest index. It is seen that phenols also play an important role in the initiation and development of nodules.     

Phenolic Compound Utilization by the Soft Rot Fungus Lecythophora hoffmannii

Nine phenolic compounds were metabolized by the soft rot fungus Lecythophora hoffmannii via protocatechuic acid and subsequently cleaved by protocatechuate 3,4-dioxygenase as determined by oxygen uptake, substrate depletion, and ring cleavage analysis. Catechol was metabolized by catechol 1,2-dioxygenase. Fungal utilization of these aromatic compounds may be important in the metabolism of wood decay products.     

Evidence of two pathways for the metabolism of phenol by Aspergillus fumigatus

Aspergillus fumigatus (ATCC 28282), a thermotolerant fungus, has been shown to be capable of growth on phenol as the sole carbon and energy source. During growth of the organism on phenol, catechol and hydroquinone accumulated transiently in the medium; cells grown on phenol oxidised these compounds without a lag period. Two different routes operating simultaneously, leading to different ring-fission substrates, are proposed for the metabolism of phenol. In one route, phenol undergoes ortho-hydroxylation to give catechol, which is then cleaved by an intradiol mechanism leading to 3-oxoadipate. In the other route, phenol is hydroxylated in the para-position to produce hydroquinone, which is then converted into 1,2,4-trihydroxybenzene for ring fission by ortho-cleavage to give maleylacetate. Cell-free extracts of phenol-grown mycelia were found to contain enzymic activities for the proposed steps. Two ring-fission dioxygenases, one active towards 1,2,4-trihydroxybenzene, but not catechol, and one active towards both ring-fission substrates, were separated by FPLC. Succinate-grown mycelia did not oxidise any of the intermediates until a clear lag period had elapsed and did not contain any of the enzymic activities for phenol metabolism.     

Effect of alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanologenic Escherichia coli

Lignocellulose can be readily hydrolyzed into a mixture of sugars using dilute mineral acids. During hydrolysis, a variety of inhibitors are also produced which include aromatic alcohols from lignin and furfuryl alcohol from pentose destruction. Seven compounds were investigated individually and in binary combinations (catechol, coniferyl alcohol, furfuryl alcohol, guaiacol, hydroquinone, methylcatechol, and vanillyl alcohol). Aromatic alcohols and furfuryl alcohol inhibited ethanol production from xylose in batch fermentations primarily by inhibiting the growth of Escherichia coli LY01, the biocatalyst. The toxicities of these compounds were directly related to their hydrophobicity. Methylcatechol was the most toxic compound tested (MIC = 1.5 g/L). In binary combination, the extent of growth inhibition was roughly additive for most compounds tested. However, combinations with furfuryl alcohol and furfural (furaldehyde) appear synergistic in toxicity. When compared individually, alcohol components which are formed during hemicellulose hydrolysis are less toxic for growth than the aldehydes and organic acids either on a weight basis or a molar basis.     

Contribution of Bioactive Compounds to the Antioxidant Capacity of the Edible Mushroom Neolentinus lepideus

Neolentinus lepideus is a fungus consumed by rural communities in Central America and Asia due to its rich flavor; however, little information on its chemical composition is available. With this in mind, the objective of this work was to determine the content of vitamin E and C, ergosterol, and phenolic compounds of this fungus, as well as its antioxidant capacity. The quantified bioactive compounds were two isoforms of vitamin E, highlighting α-tocopherol (3370.35 mg/100 g dry weight, DW) and ergosterol (11.70 mg/100 g DW). The total phenolic content was 164.80 mg gallic acid equivalents/100 g, and nine phenolic compounds were identified (protocatechuic, p-hydroxybenzoic, caffeic, vanillic, ferulic, salicylic, p-anisic, trans-cinnamic acids, and scopoletin). The highest antioxidant capacity was detected in the lipophilic extract with TEAC (27688 μmoles Trolox equivalents/100 g). These results suggest that lipophilic compounds are among the main bioactive compounds in N. lepideus, and they might exhibit the highest radical scavenging properties in non-polar extracts.     

Demonstration of phenolic compounds in plant tissues by an osmium-iodide postfixation procedure

A simple procedure to stain phenols in plant tissues is described. Postfixation with an aqueous solution prepared by mixing 2 cc of 2% osmium tetroxide and 8 cc of 3% potassium iodide yields brilliant visualization of phenol-containing vacuoles in different tissues of plants (e.g., coffee, oak, tobacco and spruce) bearing high concentration of phenolic compounds. Areas bearing phenols become dark gray to black. Chemical experiments demonstrate that osmium-potassium iodide (Os-KI) mixture reacts rapidly with several naturally occurring plant phenols, developing black solutions from which black solids precipitate. Phenols containing omicron-dihydroxy groups react with Os-KI solution more rapidly than other structurally different phenols. Therefore, omicron-dihydroxy units in an aromatic ring seem to function as primary sites of reactivity with the osmium-iodide complexes.     

Effect of methyl jasmonate (MeJA) on biochemical responses of wheat seedlings infected by Fusarium culmorum

Fusarium culmorum is a ubiquitous soil-borne fungus which is able to cause foot and root rot on different small-grain cereals, in particular wheat and barley. Wheat (Triticum aestivum L.) is among the oldest and most extensively grown of all crops. Systemic acquired resistance (SAR) is a pathogen-induced disease resistance response in plants that is characterized by broad spectrum disease control. SAR is an important component of the disease resistance repertoire of plants. The effect of the resistance inducer, methyl jasmonate (MeJA), was tested by seed-soaking to determine whether this resistance inducer controlled root rot of wheat caused by F. culmorum. MeJA is an inducing disease resistance in a number of dicotyledonous and monocotyledonous plant species. This research has been conducted to determine the effect of MeJA seed treatment on chlorophyll a, chlorophyll b, total protein, peroxidase (POD) enzyme, phenolic compounds and phenylalanine ammonia lyase (PAL) factors. MeJA, which is an important chemical inducer of SAR, can activate resistance to pathogen infection in commercial crops. Seeds treated with MeJA showed an elevation in the total chlorophyll, carotenoid and activity of defence-related enzymes, POD and PAL.     

Effect of lignin-derived phenolic monomers on the growth of the edible mushrooms Lentinus edodes,Pleurotus sajor-caju and Volvariella volvacea

Pleurotus sajor-caju was generally more tolerant to lignin-related phenolic monomers and tannin derivatives than Lentinus edodes and the straw mushroom, Volvariella volvacea. Several phenols, at up to 5 mM, enhanced mycelial growth of P. sajor-caju. No clear pattern was evident when the effects of phenols and tannins on the growth of V. volvacea and L. edodes were compared, but the lower concentrations of 4-hydroxybenzaldehyde and vanillin which were tested were markedly more toxic to the straw mushroom. The distribution of phenolic monomers and tannin derivatives in the agricultural wastes used for mushroom cultivation may be an important growth determinant. However, the differences in the growth inhibition profiles of L. edodes, P. sajor-caju and V. volvacea suggest that, alone, the effect of these compounds on fungal growth is unlikely to account for the varying abilities of the three mushroom species to grow and fruit on a particular lignocellulosic substrate.     

Biodegradation of mixed phenolic compounds by Aspergillus awamori NRRL 3112

The ability of the fungus Aspergillus awamori NRRL 3112 to degrade mixtures of some common phenolic compounds, namely phenol, catechol, 2,4-dichlorphenol and 2,6-dimethoxyphenol was investigated in the present study. For all combinations in which dichlorophenol was incorporated, it took equal time for the nearly complete degradation of the compound—4 days. Phenol was decomposed almost completely (99.5%) in a combination with dimethoxyphenol, to a lesser extent (88%) in a combination with catechol and to the least degree (25%) in the presence of 2,4-dichlorophenol. Catechol experienced a more substantial biotransformation (64%) when mixed with phenol and weaker (45%)—in a combination with dichlorophenol. 2,6-Dimethoxyphenol was better decomposed (69%) in mixtures containing phenol, while its biodegradation in a combination with 2,4-dichlorophenol was considerably poor (only 5%).     

Degradation of Benzene,Toluene and Xylene by Pseudomonas aeruginosa Engineered with the Vitreoscilla Hemoglobin Gene

This study concerns the potential use of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene for the degradation of important harmful aromatic compounds such as benzene, toluene, and xylene (BTX). The use of these compounds by both strains was determined as the production of cell mass (viable cell number) in a minimal medium containing any one of the BTX compounds as the sole carbon and energy source. Furthermore, the BTX degradation capability of both strains was monitored by measuring the production of 3‐methylcatechol, a common intermediate. For the cells of the logarithmic phase, which were grown at high aeration/high agitation or low aeration/low agitation, the engineered strain showed a better growth rate than the host strain. With the benzene in the medium, the recombinant strain exhibited a higher (up to 4‐fold) cell density than the parental wild‐type strain at this phase. In contrast, regarding the cells of the late stationary phase under high aeration/high agitation conditions, the host strain had generally higher viable cell numbers than the recombinant strain. At this phase this difference was, however, less significant under the conditions of low aeration/low agitation. Similarly, in toluene containing medium (at high aeration/high agitation) the recombinant strain showed a higher cell density which was from a 15‐fold to almost one order of magnitude greater than its parental strain during the logarithmic phase where the cell density of P. aeruginosa remained nearly constant. Contrary to the results with benzene and toluene, both strains exhibited similar growth characteristics when they were grown in the presence of xylene. The positive effect of the oxygen uptake by the recombinant system on the BTX metabolizing activity was also apparent in a high accumulation of 3‐methylcatechol in the cultures of the recombinant strain. At certain points of incubation, the hemoglobin expressing strain showed a significantly (p < 0.05) higher 3‐methylcatechol accumulation than the host strain. These results demonstrated the possible potential of the Vitreoscilla hemoglobin as an efficient oxygen uptake system for the bioremediation of some compounds of environmental concern.     

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.     

Wine phenolic compounds influence the production of volatile phenols by wine-related lactic acid bacteria

Aims: To evaluate the effect of wine phenolic compounds on the production of volatile phenols (4‐vinylphenol [4VP] and 4‐ethylphenol [4EP]) from the metabolism of p‐coumaric acid by lactic acid bacteria (LAB). Methods and Results: Lactobacillus plantarum, Lactobacillus collinoides and Pediococcus pentosaceus were grown in MRS medium supplemented with p‐coumaric acid, in the presence of different phenolic compounds: nonflavonoids (hydroxycinnamic and benzoic acids) and flavonoids (flavonols and flavanols). The inducibility of the enzymes involved in the p‐coumaric acid metabolism was studied in resting cells. The hydroxycinnamic acids tested stimulated the capacity of LAB to synthesize volatile phenols. Growth in the presence of hydroxycinnamic acids, especially caffeic acid, induced the production of 4VP by resting cells. The hydroxybenzoic acids did not significantly affect the behaviour of the studied strains. Some of the flavonoids showed an effect on the production of volatile phenols, although strongly dependent on the bacterial species. Relatively high concentrations (1 g l^?1) of tannins inhibited the synthesis of 4VP by Lact. plantarum. Conclusions: Hydroxycinnamic acids were the main compounds stimulating the production of volatile phenols by LAB. The results suggest that caffeic and ferulic acids induce the synthesis of the cinnamate decarboxylase involved in the metabolism of p‐coumaric acid. On the other hand, tannins exert an inhibitory effect. Significance and Impact of the Study: This study highlights the capacity of LAB to produce volatile phenols and that this activity is markedly influenced by the phenolic composition of the medium.     

Antioxidant Activity and Phytopathogenic Control of Extracts and Fraction from Struthanthus calophyllus A.C.Sm. (Loranthaceae)

Phytopathogenic microorganisms cause oxidative stress in host plants, thus affecting agricultural crops. Such stress could be controlled by antioxidant compounds from parasitic plants, given their antioxidant power. This article reports an evaluation of Struthanthus calophyllus antimicrobial activity and antioxidant mechanism by testing different polarity extracts. Antimicrobial activity was evaluated against phytopathogens bacteria (e. g., Erwinia, Pectobacterium, Xanthomonas) using the agar diffusion method. Pectobacterium and Xanthomonas presented growth inhibition zones similar to streptomycin control. Antioxidant activity was determined by measuring total phenol content, DPPH⋅, and ABTS⋅⁺ radicals-scavenging inhibition percentage (IP). Most polar extracts presented 76,9–95,9 % IP, which correlates with its phenolic content. Besides, Nuclear Magnetic Resonance in a V14-1 sub-fraction from stem ethanolic extract, chose one for highest yield percentage, highest metabolites presence, and antimicrobial activity, showed triterpenic compounds mixture (α-amyrin, β-amyrin and 24-methylenecycloartanol). Findings in this study are among the first reports for S. calophyllus, even the chemical characterization, that confirm its strong antioxidant and antibacterial activities. Further molecular composition research about parasitic plants could show how unknown compounds may combat pathogenic microorganisms.     

Establishment and growth characteristics of Glycyrrhiza glabra hairy root cultures

Hairy root cultures of Glycyrrhiza glabra producing considerable amounts of phenolic compounds were successfully established by using Agrobacterium strain C58C18(pRT GUS 104). The effect of phosphate, ammonia, nitrate and ferric-EDTA concentrations of culture medium on growth and total phenolics production of the cultures were studied. By employing statistical experimental design and linear regression analysis an improved B5 medium (B50-M) could be developed. When cultivating G. glabra hairy roots in B50-M medium we were able to obtain 9 g dried roots/l in 25 days which was twice as much as when using the initial B50 medium. According to tentative analyses the cultures did not contain glycyrrhyzin, but they produced liquiritigenin and isoliquiritigenin. The production of total phenolic substances (mg g^-1 dw) was higher in the improved medium resulting in significantly higher volumetric productivity (mg phenolic compounds l^-1). This will further enable the extraction and identification of the phenolic compounds produced by the cultures.     

Enzyme activity of the phenylpropanoid pathway as a response to apple scab infection

The study was performed on apple trees, ‘Golden Delicious' cv., which is a scab-susceptible cultivar. The phenolic content of apple fruit was determined in different parts of the peel. The phenolic compounds were analysed in the scab spot, in the tissue around the spot and in the healthy tissue. We determined the concentration of various phenolic compounds and related enzyme activities. Infection with the Venturia inaequalis fungus enhanced the metabolism of phenolic compounds at the scab spot, around the spot and in healthy peel. Compared with the healthy tissue and the tissue around the spot, the scab spot showed higher enzyme activity for all tested enzymes, except for dihydrochalcone 2′-O-glucosyltransferase, which had lower activity in the scab spot. In comparison to the healthy peel, the scab spot showed up to 3.4 times more hydroxycinnamic acids, up to 1.1 times more dihydrochalcones and up to 1.4 times more flavan-3-ols. In contrast, the healthy peel showed up to 1.6 times more flavonols than the scab spot.