Oxidoreductases provide a more generic response to metallic stressors (Cu and Cd) than hydrolases in soil fungi: new ecotoxicological insights

The present study investigates the effect of metals on the secretion of enzymes from12 fungal strains maintained in liquid cultures. Hydrolases (acid phosphatase, β-glucosidase, β-galactosidase, and N-acetyl-β-glucosaminidase) and ligninolytic oxidoreductases (laccase, Mn, and lignin peroxidases) activities, as well as biomass production, were measured in culture fluids from fungi exposed to Cu or Cd. Our results showed that all fungi secreted most of the selected hydrolases and that about 50 % of them produced a partial oxidative system in the absence of metals. Then, exposure of fungi to metals led to the decrease in biomass production. At the enzymatic level, Cu and Cd modified the secretion profiles of soil fungi. The response of hydrolases to metals was contrasted and complex and depended on metal, enzyme, and fungal strain considered. By contrast, the metals always stimulated the activity of ligninolytic oxidoreductases in fungal strains. In some of them, oxidoreductases were specifically produced following metal exposure. Fungal oxidoreductases provide a more generic response than hydrolases, constituting thus a physiological basis for their use as biomarkers of metal exposure in soils.     

Secretion profiles of fungi as potential tools for metal ecotoxicity assessment: a study of enzymatic system in Trametes versicolor

The relationship between the expression of extracellular enzymatic system and a metal stress is scarce in fungi, hence limiting the possible use of secretion profiles as tools for metal ecotoxicity assessment. In the present study, we investigated the effect of Zn, Cu, Pb and Cd, tested alone or in equimolar cocktail, on the secretion profiles at enzymatic and protein levels in Trametesversicolor. For that purpose, extracellular hydrolases (acid phosphatase, β-glucosidase, β-galactosidase and N-acetyl-β-glucosaminidase) and ligninolytic oxidases (laccase, Mn-peroxidase) were monitored in liquid cultures. Fungal secretome was analyzed by electrophoresis and laccase secretion was characterized by western-blot and mass spectrometry analyses. Our results showed that all hydrolase activities were inhibited by the metals tested alone or in cocktail, whereas oxidase activities were specifically stimulated by Cu, Cd and metal cocktail. At protein level, metal exposure modified the electrophoretic profiles of fungal secretome and affected the diversity of secreted proteins. Two laccase isoenzymes, LacA and LacB, identified by mass spectrometry were differentially glycosylated according to the metal exposure. The amount of secreted LacA and LacB was strongly correlated with the stimulation of laccase activity by Cu, Cd and metal cocktail. These modifications of extracellular enzymatic system suggest that fungal oxidases could be used as biomarkers of metal exposure.     

Influence of a soil enzyme on iron-cyanide complex speciation and mineral adsorption

Cyanide is commonly found as ferrocyanide [Fe(II)(CN)(6)](-4) and in the more mobile form, ferricyanide [Fe(III)(CN)(6)](-3) in contaminated soils and sediments. Although soil minerals may influence ferrocyanide speciation, and thus mobility, the possible influence of soil enzymes has not been examined. In a series of experiments conducted under a range of soil-like conditions, laccase, a phenoloxidase enzyme derived from the fungi Trametes versicolor, was found to exert a large influence on iron-cyanide speciation and mobility. In the presence of laccase, up to 93% of ferrocyanide (36-362ppm) was oxidized to ferricyanide within 4h. No significant effect of pH (3.6 and 6.2) or initial ferrocyanide concentration on the extent or rate of oxidation was found and ferrocyanide oxidation did not occur in the absence of laccase. Relative to iron-cyanide-mineral systems without laccase, ferrocyanide adsorption to aluminum hydroxide and montmorillonite decreased in the presence of laccase and was similar to or somewhat greater than that of ferricyanide without laccase. Laccase-catalyzed conversion of ferrocyanide to ferricyanide was extensive though up to 33% of the enzyme was mineral-bound. These results demonstrate that soil enzymes can play a major role in ferrocyanide speciation and mobility. Biotic soil components must be considered as highly effective oxidation catalysts that may alter the mobility of metals and metal complexes in soil. Immobilized enzymes should also be considered for use in soil metal remediation efforts.     

Removal of estrogenic activity of natural steroidal hormone estrone by ligninolytic enzymes from white rot fungi

Natural steroidal hormone estrone (E1) was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. E1 decreased by 98% after 5 d of treatment and the activities of ligninolytic enzymes, manganese peroxidase (MnP) and laccase, were detected during treatment, which suggested that the disappearance of E1 is related to ligninolytic enzymes produced extracellularly by white rot fungus. Therefore, E1 was treated with MnP and laccase prepared from the culture of white rot fungi. HPLC analysis demonstrated that E1 disappeared completely in the reaction mixture after 1 h of treatment with either MnP or laccase. Using the yeast two-hybrid assay system, it was also confirmed that both enzymatic treatments completely removed the estrogenic activity of E1 after 2 h. These results strongly suggest that ligninolytic enzymes are effective in removing the estrogenic activity of E1.     

Laccase activity in soils: considerations for the measurement of enzyme activity

Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing enzymes that catalyze the oxidative conversion of a variety of chemicals, such as mono-, oligo-, and polyphenols and aromatic amines. Laccases have been proposed to participate in the transformation of organic matter and xenobiotics as well as microbial interactions. Several laccase assays have been proposed and used in soils. Here, we show that the optimal pH conditions for the laccase substrates 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, pH 3-5), 2,6-dimethoxyphenol (4-5.5), L-3,4-dihydroxyphenylalanine (DOPA; 4-6), guaiacol (3.5-5), 4-methylcatechol (3.5-5), and syringaldazine (5.5-7.0) are similar between purified laccases from Trametes versicolor and Pyricularia sp. and soil extracts; the substrate affinities of purified enzymes (K(M)) and soil extracts were also similar. The laccase assays showed specificity overlap with tyrosinase and ligninolytic peroxidases when hydrogen peroxide is present. The ABTS oxidation assay is able to reliably detect the presence of 13.5 pg mL(-1) or 0.199×10(-12) mol mL(-1) of T. versicolor laccase, which is three times more sensitive than the 2,6-dimethoxyphenol-based assay and more than 40 times more sensitive than any of the other assays. The low molecular mass soil-derived compounds and the isolated fulvic and humic acids influence the laccase assays and should be removed from the soil extracts before measurements of the enzyme activity are performed.     

Fungal laccase-catalyzed degradation of hydroxy polychlorinated biphenyls

Hydroxy polychlorinated biphenyls (hydroxy PCBs) are toxic metabolites of PCBs. Their toxicity such as strong endocrine disruption demands effective remediation methods. Laccases from Trametes versicolor and Pleurotus ostreatus were tested to degrade hydroxy PCBs. Optimum pHs for both enzymes were around 4.0. Laccase from T. versicolor degrades hydroxy PCBs more rapidly than that from P. ostreatus. The enzymatic activities remained little changes in up to 10% organic solvents, but decreased rapidly in more than 10% acetone, acetonitrile or DMSO. Degradation rate constants decreased with increase of chlorination and no degradation was observed with tetra-, penta- and hexa-chloro hydroxy PCBs in non-mediated reactions. However, the tetra- to hexa-chloro hydroxy PCBs were degraded by laccase from T. versicolor in the presence of the mediator 2,2,6,6-tetramethylpiperidine-N-oxy radical. The other mediators, 4-ethyl-2-methoxyphenol, 2,2'-azino-bis(3-ethylbenzthiazoline sulfonic acid) diammonium salt and 1-hydroxybenzotriazole and humic acid, also enhanced degradation of all the hydroxy PCBs except 4-hydroxy-2',3,3',4',5,5'-hexachlorobiphenyl. The results showed that 3-hydroxy biphenyl was more resistant to laccase degradation than 2- or 4-hydroxy analogues. Significant linear-correlations (coefficient of determination, r2 = 0.9097 and 0.8186 for laccases from P. ostreatus and T. versicolor, respectively) were found between the ionization potentials and the removal rate constants of hydroxy PCBs.     

Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds

Due to the numerous biotechnological applications of laccase enzyme, it is essential to know the influence of different agents usually present in the natural environment on its enzymatic action, especially for in situ treatment technologies. In the present work, a simple and rapid method to determine the inhibitory or inducer effect of different compounds on laccase activity was developed. The compounds tested were copper-chelating agents and heavy metals. It was found that using syringaldazine as a substrate, all copper-chelating agents (except EDTA) highly inhibited laccase activity (around 100%) at an inhibitor concentration lower than 20 mM. Moreover, 40% of inhibition, which was detected at a concentration of 20 mM for both Cd(2+) and Cu(2+) increased with concentration until nearly complete inhibition at 80 mM.     

Mandarin peelings: the best carbon source to produce laccase by static cultures of Trametes pubescens

In the present study, we investigated the effect of different carbon sources (glucose, glycerol and ground mandarin peelings) on laccase production by Trametes pubescens grown on stainless steel sponges under static conditions. The cultures with ground mandarin peelings gave the highest laccase activities, showing values of about 100 U l(-1). This is a very interesting result, since mandarin peelings are common agricultural wastes in some regions such as Mediterranean and Asiatic countries. Therefore, their reutilisation, besides reducing medium cost, also helps to solve the pollution problems caused by their disposal. Also, we studied the effect of supplementing the culture medium with different potential laccase-inducing compounds (ABTS, Tween 20, soya oil, Malaquite Green, Cu(2+), tannic acid) on laccase production. Soya oil was the best inducer of laccase activities, attaining values 4-fold higher than those obtained in the reference cultures.     

Comparative study of immobilized Trametes versicolor laccase on nanoparticles and kaolinite

Laccase from Trametes versicolor was immobilized on nanoparticles and kaolinite by physical adsorption or chemical covalence in which the supporters were activated by cross-linked with glutaraldehyde. Thermal and pH stabilities of immobilized laccase on these different supporters were compared. The degradation efficiencies of these immobilized laccases on oxidation of benzo[a]pyrene (BaP) were also compared. The results showed that the immobilized laccases on nanoparticles were more stable in resisting pH and thermal changes. After 48h oxidation, laccase immobilized on kaolinite using the covalent coupling method showed a higher efficiency of oxidation with the BaP residue of 23% in the presence of 1mM HBT and with BaP residue of 37% in 1mM ABTS as the mediator. The results also exhibited a significant inhibition by 1% surfactant Tween 80. According to the HPLC analysis, the oxidation products including 1,6-benzo[a]pyrene quinone, 3,6-benzo[a]pyrene quinone and 6,12-benzo[a]pyrene quinone were identified.     

Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor

Some researches studied the removal of steroid estrogens by enzymatic treatment, however none verified the residual estrogenicity after the enzymatic treatment at environmental conditions. In this study, the residual estrogenic activities of the key natural and synthetic steroid estrogens were investigated following enzymatic treatment with horseradish peroxidase (HRP) and laccase from Trametes versicolor. Synthetic water and municipal wastewater containing environmental concentrations of estrone, 17beta-estradiol, estriol, and 17alpha-ethinylestradiol were treated. Liquid chromatography-mass spectrometry analysis demonstrated that the studied steroid estrogens were completely oxidized in the wastewater reaction mixture after a 1-h treatment with either HRP (8-10 U ml(-1)) or laccase (20 U ml(-1)). Using the recombinant yeast assay, it was also confirmed that both enzymatic treatments were very efficient in removing the estrogenic activity of the studied steroid estrogens. The laccase-catalyzed process seemed to present great advantages over the HRP-catalyzed system for up-scale applications for the treatment of municipal wastewater.     

Cadmium and lead bioavailability and their effects on polycyclic aromatic hydrocarbons biodegradation by spent mushroom substrate

Bioremediation of mixed metal–organic soil pollution constitutes a difficult task in different ecosystems all around the world. The aims of this work are to determine the capacity of two spent mushroom substrates (Agaricus bisporus and Pleurotus ostreatus) to immobilize Cd and Pb, to assess the effect of these metals on laccase activity, and to determine the potential of spent A. bisporus substrate to biodegrade four polycyclic aromatic hydrocarbons (PAH): fluorene, phenanthrene, anthracene, and pyrene, when those toxic heavy metals Cd and Pb are present. According to adsorption isotherms, spent P. ostreatus and A. bisporus substrates showed a high Pb and Cd adsorption capacity. Pb and Cd interactions with crude laccase enzyme extracts from spent P. ostreatus and A. bisporus substrates showed Cd and Pb enzyme inhibition; however, laccase activity of A. bisporus presented lower inhibition. Spent A. bisporus substrate polluted with PAH and Cd or Pb was able to biodegrade PAH, although both metals decrease the biodegradation rate. Spent A. bisporus substrate contained a microbiological consortium able to oxidize PAH with high ionization potential. Cd and Pb were immobilized during the bioremediation process by spent A. bisporus substrate. Consequently, spent A. bisporus substrate was adequate as a multi-polluted soil bioremediator.     

Comparative studies of fungal degradation of single or mixed bioaccessible reactive azo dyes

A screening using several fungi (Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Aureobasidium pullulans) was performed on the degradation of syringol derivatives of azo dyes possessing either carboxylic or sulphonic groups, under optimized conditions previously established by us. T. versicolor showed the best biodegradation performance and its potential was confirmed by the degradation of differently substituted fungal bioaccessible dyes. Enzymatic assays (lignin peroxidase, manganese peroxidase, laccase, proteases and glyoxal oxidase) and GC-MS analysis were performed upon the assay obtained using the most degraded dye. The identification of hydroxylated metabolites allowed us to propose a possible metabolic pathway. Biodegradation assays using mixtures of these bioaccessible dyes were performed to evaluate the possibility of a fungal wastewater treatment for textile industries.     

Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter

Marasmius quercophilus is a white-rot fungus involved in carbon recycling in Mediterranean ecosystems because of its laccase production. Here we described the effect of metal ions and halide salts, on laccase activity in order to point out the action of such environmental pollutants on this enzyme of major importance. Furthermore we tested organic solvent effects on laccase reaction since reaction mixture including solvent can be used in the transformation of xenobiotics. In the case of metal ions, we found that chloride ions were responsible for inhibition while CuSO(4) and MnSO(4) enhanced laccase activity. When halides were tested, we showed the following degree of inhibition: F(-)>Cl(-)>Br(-). Furthermore we found that I(-) was oxidized by laccase with I(2) as the product of the reaction. With ABTS, 50% of the laccase activity remains for solvent concentration ranging from 40% to 60% depending on the solvent used while with syringaldazine solvent concentration ranged from 50% to 70%. The organic solvent effects observed were probably a result of enzyme denaturation and of both enhancement of oxidised product solubilisation and of substrate solubilisation (for syringaldazine). These results show that laccase from M. quercophilus is not rapidly inhibited by certain environmental pollutants which sustains its role in carbon turnover under pertubation. However the strong effect of chloride ion on laccase activity should be further investigated with in situ studies since this could drastically influence carbon recycling in litters from Mediterranean littoral locations.     

Influence of redox mediators and metal ions on synthetic acid dye decolourization by crude laccase from Trametes hirsuta

In this paper, the effect of redox mediators on synthetic acid dye decolourization (Sella Solid Red and Luganil Green) by laccase from Trametes hirsuta cultures has been investigated. All the redox mediators tested, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT) and Remazol Brilliant Blue R (RBBR), led to higher activities than those obtained without mediators addition showing the suitability of the laccase/mediator system (LMS) in the decolourization of acid dyes. HBT was by far the most effective mediator, showing a decolourization percentage of 88% in 10 min for Sella Solid Red and of 49% in 20 min for Luganil Green. On the other hand, the stability of laccase against several metal ions, normally found in textile wastewater, was assessed. Laccase was stable at a concentration of 1mM for 7d against all the metal ions tested except for Zn+2, CrO4(-2), Cd+2, Cr2O7(-2), Fe+2, Cu+2 and especially Hg+2. When the concentration was increased to 10mM laccase stability decreased against all the metals assayed, in particular against Fe+2. In addition, the effect of metal ions on the decolourization process was also studied. It was found that Hg+2 inhibited the dye decolourization process, being the presence of HBT absolutely required for dye decolourization.     

Reusing ethyl acetate and aqueous exhausted fractions of dry olive mill residue by saprobe fungi

Some saprobe fungi (Phlebia radiata, Trametes versicolor, Coriolopsis rigida, Pycnoporus cinnabarinus, Fomes sclerodermus or Pleurotus pulmonarius) were able to bioconvert the ethyl acetate fraction (DEAF) and the corresponding aqueous exhausted fraction (EAF) of dry olive mill residue (DOR), reducing their phytotoxicity on Lepidium sativum seeds. Large amount of hydroxytyrosol together with other eight monomeric phenols were found in the native DEAF fraction, which represents a good source of antioxidants. P. radiata, T. versicolor and F. sclerodermus caused an effective phytotoxicity reduction of EAF in the concentration range of 25-3 gl(-1). In particular, in the range between 12.5 and 3 gl(-1), the EAF samples inoculated with P. radiata and F. sclerodermus surprisingly stimulated the germinability of L. sativum, suggesting their use as a potential biofertilizer. This is the first report which showed the bioconversion of the above fractions in shorter time with respect to the previous findings concerning DOR. The possible implications of laccase in the decrease of DEAF and EAF phytotoxicity was also discussed.     

Enhancing Diatom Growth by Using Zeolites to Change Seawater Composition (4 pp)

- Aims and Scope. The purpose of this study was to test whether two zeolites produced synthetically (products of zeolitic nature, PZN) could influence either the yield of a diatom culture or the chemical changes in the cultures. For this purpose, Phaeodactylum tricornutum was used as test organism in a culture medium of natural seawater enriched with N and P having negligible amounts of ammonia. Methods The PZN ZEBEN-06 and ZESTEC-56 were used in parallel experiments. The composition of trace metals and organic compounds with chelate activity in the culture media, were determined by anodic or cathodic stripping voltammetry. The impact of leaching silicon on the algal yield was evaluated by comparing the growth in the presence or absence of PZN, in seawater enriched or not-enriched with silicon. Results and Conclusions Both PZN significantly promoted the algal yield even in the absence of added silicon, a limiting nutrient for diatom growth. The PZN acted as a silicon buffer while providing a source of silicon required for growth. In addition, PZN released into the seawater small but significant amounts of the limiting micro-nutrient manganese (its concentration doubled during the experiments), while simultaneously removing relatively high quantities of zinc from the seawater. The presence of PZN inhibited the releasing of chelated compounds. These changes (sorption/desorption) caused by the PZN in the concentrations in the solutions used as culture media of P. tricornutum were probably responsible for the differences in both the diatom and exudation observed in the tested cultures.     

Migration of heavy metals and migration-degradation of phenanthrene in soil using electro kinetic-laccase combined remediation system

Abstract

In order to solve the problem of heavy metal-organic compound soil pollution, in this paper, we developed a highly efficient electro kinetic-laccase combined remediation (EKLCR) system. The results showed that the EKLCR system had an obvious migration effect on heavy metals (copper and cadmium) and good migration-degradation effect on phenanthrene. The migration rates of copper and cadmium were 48.3% and 40.3%, respectively. Especially, with the presence of laccase, the removal rate of phenanthrene on Cu²⁺-contaminated soil was higher than that of Cd²⁺-contaminated soil due to the significant effect of heavy metals on the enzymatic activity of laccase. The average migration-degradation rate of phenanthrene by EKLCR system was 45.4%. Finally, gas chromatography-mass spectrometry (GC/MS) was used to analyze the degradation intermediates of phenanthrene in the soil, which included 9,10-Phenanthrenequinone, phthalic acid, and 2,2-Biphenyldicarboxylic Acid. In addition, we give the possible degradation pathways of phenanthrene, 2,2-Biphenyldicarboxylic Acid is further degraded to produce phthalic acid. The products of the phthalic acid metabolic pathway are protocatechuic acid, pyruvic acid or succinic acid, the final products of these organic acids are carbon dioxide and water.     

城市污水厂污泥替代营养盐培养海水小球藻的研究

为了城市污水厂污泥的资源化利用,对污泥替代F/2培养基培养海水小球藻(Chlorella pacifica)进行了研究。首先采用研磨-离心法制备污泥抽提液,将其与F/2培养基按体积比7∶3混合,接种微藻并在优化的条件下培养6 d,逐日测定混合液中无机氮、磷的浓度,培养结束后测定微藻生物量以及干藻中蛋白质、氨基酸和重金属含量。结果表明,将微藻在最佳条件下培养6 d后,混合液中PO34--P、NO2--N、NH4+-N和NO3--N的吸收率分别达到97.32%、96.72%、97.55%和78.13%;与F/2培养基对照组相比,混合液中微藻的生物量增加11.5%;蛋白质、必需氨基酸和非必需氨基酸的含量变化不大。另外,干藻中的重金属含量符合饲料行业标准的要求。     

Exoenzymes of Trametes versicolor can metabolize coplanar PCB congeners and hydroxy PCB

Two fractions containing the oxidase activity toward 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) were obtained using ion-exchange DEAE-Sepharose column chromatography of the culture fluid of white-rot fungus, Trametes versicolor. These two fractions can reduce the level of coplanar PCB congeners (Co-PCBs). The ABTS oxidase in the first fraction passed through the DEAE-Sepharose column. The ABTS oxidase in the second fraction was adsorbed to the column at 相似文献   

Coupling occurs before breakdown during biotransformation of Acid Blue 62 by white rot fungi

Only few data exist on the metabolites produced during the biotransformation of anthraquinonic dyes by white rot fungi (WRF). During the biotransformation of an anthraquinonic dye Acid Blue 62 (ABu62) using Pycnoporus sanguineus MUCL 41582 strain, it was previously demonstrated that the blue colour of the medium turned to red before complete dye decolourisation. To better understand the phenomenon, this study carried out ABu62 biotransformation with five different WRF strains (Coriolopsis polyzona MUCL 38443, Perenniporia ochroleuca MUCL 41114, Perenniporia tephropora MUCL 41562, P. sanguineus MUCL 38531 and Trametes versicolor MUCL 38412) and compared with P. sanguineus MUCL 41582 previously described. A multi-methodological approach (using capillary electrophoresis, mass spectrometry, HPLC, UV, NMR and IR spectroscopies) was developed to characterise the metabolites involved and monitor their apparition. Seven metabolites were commonly found with all strains, suggesting a common metabolic pathway for ABu62 biotransformation. During the first days, dimer and oligomers of the initial ABu62 molecule were observed: the main one absorbed in the 500nm region, explaining the red colour appearance of the medium. This main metabolite was made up of two molecules of ABu62 linked by an azo bond, minus a cyclohexyl moiety. After a longer incubation time, breakdown products were observed. Based on these products characterizations, a bioconversion pathway was proposed.