Production Of Lignin-modifying Enzymes Research Articles

Regulation of dye-decolorizing peroxidase gene expression in Pleurotus ostreatus grown on glycerol as the carbon source.

Dye-decolorizing peroxidases (DyPs) (E.C. 1.11.1.19) are heme peroxidases that catalyze oxygen transfer reactions similarly to oxygenases. DyPs utilize hydrogen peroxide (H2O2) both as an electron acceptor co-substrate and as an electron donor when oxidized to their respective radicals. The production of both DyPs and lignin-modifying enzymes (LMEs) is regulated by the carbon source, although less readily metabolizable carbon sources do improve LME production. The present study analyzed the effect of glycerol on Pleurotus ostreatus growth, total DyP activity, and the expression of three Pleos-dyp genes (Pleos-dyp1, Pleos-dyp2 and Pleos-dyp4), via real-time RT-qPCR, monitoring the time course of P. ostreatus cultures supplemented with either glycerol or glucose and Acetyl Yellow G (AYG) dye. The results obtained indicate that glycerol negatively affects P. ostreatus growth, giving a biomass production of 5.31 and 5.62 g/L with respective growth rates (micra; m) of 0.027 and 0.023 h-1 for fermentations in the absence and presence of AYG dye. In contrast, respective biomass production levels of 7.09 and 7.20 g/L and growth rates (μ) of 0.033 and 0.047 h-1 were observed in equivalent control fermentations conducted with glucose in the absence and presence of AYG dye. Higher DyP activity levels, 4,043 and 4,902 IU/L, were obtained for fermentations conducted on glycerol, equivalent to 2.6-fold and 3.16-fold higher than the activity observed when glucose is used as the carbon source. The differential regulation of the DyP-encoding genes in P. ostreatus were explored, evaluating the carbon source, the growth phase, and the influence of the dye. The global analysis of the expression patterns throughout the fermentation showed the up- and down- regulation of the three Pleos-dyp genes evaluated. The highest induction observed for the control media was that found for the Pleos-dyp1 gene, which is equivalent to an 11.1-fold increase in relative expression (log2) during the stationary phase of the culture (360 h), and for the glucose/AYG media was Pleos-dyp-4 with 8.28-fold increase after 168 h. In addition, glycerol preferentially induced the Pleos-dyp1 and Pleos-dyp2 genes, leading to respective 11.61 and 4.28-fold increases after 144 h. After 360 and 504 h of culture, 12.86 and 4.02-fold increases were observed in the induction levels presented by Pleos-dyp1 and Pleos-dyp2, respectively, in the presence of AYG. When transcription levels were referred to those found in the control media, adding AYG led to up-regulation of the three dyp genes throughout the fermentation. Contrary to the fermentation with glycerol, where up- and down-regulation was observed. The present study is the first report describing the effect of a less-metabolizable carbon source, such as glycerol, on the differential expression of DyP-encoding genes and their corresponding activity.

Physiological function of hydrophobin Vmh3 in lignin degradation by white-rot fungus Pleurotus ostreatus.

Hydrophobins are small-secreted proteins comprising both hydrophobic and hydrophilic parts, that can self-assemble into an amphiphilic film at the air-liquid interface. More than 20 hydrophobin genes have been estimated in the white-rot fungus Pleurotus ostreatus. In our previous studies, three hydrophobin genes were shown to be predominantly expressed under ligninolytic conditions, and only vmh3 was downregulated in both the delignification-deficient mutant Δgat1 and Δhir1 strains. Here, we focused on the function of the hydrophobin Vmh3 to clarify its physiological role in lignin degradation. When the hyphae were observed by transmission electron microscopy, deletion of vmh3 resulted in the disappearance of black aggregates at the interface between the cell wall and outer environment. Deletion of vmh3 resulted in reduced hydrophobicity when 0.2% sodium dodecyl sulfate was dropped onto the mycelial surface. These results suggest that Vmh3 functions on the cell surface and plays a major role in mycelial hydrophobization. Furthermore, the Δvmh3 strain showed a marked delay in lignin degradation on beech wood sawdust medium, while the production of lignin-modifying enzymes was not reduced. This study demonstrated, for the first time, the possible effect of hydrophobin on lignin degradation by a white-rot fungus.

Biodecolorization of Remazol Brilliant Blue–R dye by Tropical White-Rot Fungi and Their Enzymes in The Presence of Guaiacol

The ability of the tropical white-rot fungi and their enzyme to decolorize synthetic dyes was investigated. Production of lignin-modifying enzymes (LMEs) from the three new isolated fungi, namely Trametes hirsuta D7, Ceriporia sp. BIOM 3, and Cymatoderma dendriticum WM01 were observed for 9 days incubation under static condition. The results showed that the LMEs production enhanced in the present of guaiacol. T. hirsuta D7 produced only laccase (Lac), with the highest activity was 22.6 U/L on the 5th-day of the cultivation. At the same time, Ceriporia sp. BIOM 3 and C. dendriticum WM01 secreted both laccases (Lac) with the activities 0.2 U/L and 1.0 U/L, respectively, and manganese peroxidase (MnP) with the activities 0.1 U/L and 1.0 U/L, respectively. Among the fungi, T. hirsuta D7 efficiently degraded 65% Remazol Brilliant Blue–R (RBBR) dye within 72 h using the only laccase. This study shows that laccase may have a major role in synthetic dyes' decolorization process, followed by MnP and LiP.

Determination of Lignin Modifying Enzymes from Pleurotus ostreatus and Lentinus squarrosulus

Fungi play vital roles as decomposers. White rot fungi are an eco-physiological group that degrades wood by the secretion of specialized extracellular enzymes including lignin-modifying enzymes. There is growing interest in the use of extracellular enzymes for bioremediation. This study determined the Lignin Modifying Enzymes (LMEs) associated with two species of mushroom, Pleurotus ostreatus and Lentinus squarrosulus. The qualitative study was conducted using agar medium substituted with chromogenic substances to determine the production of LMEs by the species. The appearance of colour change and clearance due to reaction with chromogenic substrates were used to determine LMEs production by the fungi. The results showed that Pleurotus ostreatus and Lentinus squarrosulus tested positive by the appearance of light brown colouration, reddish-brown colouration and discolouration of media for overall polyphenol oxidase, Laccase and Peroxidase activity, respectively. The study showed that the species studied are candidates for large scale production of LMEs that can be utilized as an eco-friendly solution for bioremediation of contaminated sites.

Production of lignin-modifying enzymes by Trametes ochracea on high-molecular weight fraction of olive mill wastewater, a byproduct of olive oil biorefinery.

Production of lignin-modifying enzymes by Trametes ochracea on high-molecular weight fraction of olive mill wastewater, a byproduct of olive oil biorefinery.

Hyper-production optimization of fungal oxidative green enzymes using citrus low-cost byproduct

The use of alternative methods is necessary to improve the production of lignin-modifying enzymes (LMEs) in the biocatalysis scenario. This study demonstrates a new and robust optimization for Lac and MnP enzymes production from Pleurotus sajor-caju induced by pulp wash citrus byproduct. The optimal values determined through response surface methodology (RSM) and artificial neural network coupled to the genetic algorithm (ANN-GA) were agitation rate (180 rpm) and pH (5.5), with a temperature of 28 ºC, after 8 days of incubation. The maximum production of Lac and MnP obtained in these conditions using RSM was 307,379.91 IU/L (R2 = 0.9566) and 11,890.20 IU/L (R2 = 0.9932), respectively. However, ANN-GA predicted a maximum production of 204,486.96 IU/L and 36,081.02 UI/L, with R2 of 0.9903. Thus, under optimized conditions, the agroindustrial pulp wash residue emerges as a promising substrate in the production of LMEs, and therefore, its biotechnological viability enables efficient and sustainable production of bioproducts.

Production of lignin-modifying enzymes by Trametes ochracea on high-molecular weight fraction of olive mill wastewater, a byproduct of olive oil biorefinery

The high-molecular weight fraction of olive mill wastewater (HMW-OMW), a byproduct of olive oil biorefinery, was used at the reactor level as the basal medium for production of laccase and Mn-dependent peroxidase (MnP) by Trametes ochracea. Three reactor systems, namely stirred tank reactors equipped with either Rushton turbines or marine impeller and draft tube (STR and STR-MD, respectively) and an air-lift reactor (ALR) were compared for this purpose. Although inocula were supplied as intact pellets, in both STR-based systems fungal growth evolved rapidly into a dispersed form while the ALR enabled the maintenance of the pellet growth mode. STR was deemed to be the most promising system since it best supported the production MnP activity on the HMW-OMW-based medium and its performance in laccase production did not differ from that observed with the STR-MD. Among the stirring regimes considered (250, 400, 500 and 600 rpm), the best production in the STR was observed at 500 rpm and 1.0 vvm for both laccase (8850 ± 270 IU L−1 on day 15) and MnP (17,027.4 ± 87.2 IU L-1 on day 13). When the inocula were supplied to the STR in homogenized form, the MnP production peak (16,856 ± 1070 IU L-1) was attained 8 days earlier than the previous condition and that of laccase was nearly doubled (14,967 ± 907 IU L-1). When compared with literature data, T. ochracea MnP production and productivity on the HMW-OMW-based medium were the highest reported for a wild-type fungal strain.

Efficient production of lignin-modifying enzymes and phenolics removal in submerged fermentation of olive mill by-products by white-rot basidiomycetes

The ability of seven distinct species of white-rot basidiomycetes (WRB) to produce lignin-modifying enzymes (LME) and reduce high phenolic content was assessed during submerged fermentation of oil mill by-products. Cerrena unicolor strains appeared to be the outstanding producers of laccase (56.3–78.5 U mL−1) and MnP (1.03–2.76 U mL−1) in the fermentation of olive pomace (OP), and laccase (26.9–93.8 U mL) in the fermentation of olive tree sawdust. Diluted 50% olive mill wastewater (OMW) can be used as a nutrient medium for laccase and MnP production but its supplementation with additional carbon and nitrogen sources and Cu2+ and Mn2= promoted the 3-, 28- and 18-fold increase of C. unicolor biomass and volumetric laccase and MnP activities, respectively, as well as 3- and 17-fold increase of Pleurotus ostreatus biomass and laccase activity, respectively. The tested fungi successfully reduced the phenolics content in the submerged fermentation of OP (72.0–81.3%) and OMW (62.4–83.5%). The laccase role in the OP phenolics reduction was proved through the treatment of 10% (wet weight) OP with commercial and lab-generated laccases preparations and reduction of total phenols by 66–70% during 24 h incubation. This work indicated that the olive mill by-products fermentation by individual substrate-specific WRB is an economically feasible and promising alternative for the simultaneous production of LME, cellulase, xylanase and detoxified materials suitable for further exploitation in biotechnological and agronomic applications.

Physiological Peculiarities of Lignin-Modifying Enzyme Production by the White-Rot Basidiomycete Coriolopsis gallica Strain BCC 142.

Sixteen white-rot Basidiomycota isolates were screened for production of lignin-modifying enzymes (LME) in glycerol- and mandarin peel-containing media. In the synthetic medium, Cerrena unicolor strains were the only high laccase (Lac) (3.2–9.4 U/mL) and manganese peroxidase (MnP) (0.56–1.64 U/mL) producers while one isolate Coriolopsis gallica was the only lignin peroxidase (LiP) (0.07 U/mL) producer. Addition of mandarin peels to the synthetic medium promoted Lac production either due to an increase in fungal biomass (Funalia trogii, Trametes hirsuta, and T. versicolor) or enhancement of enzyme production (C. unicolor, Merulius tremellosus, Phlebia radiata, Trametes ochracea). Mandarin peels favored enhanced MnP and LiP secretion by the majority of the tested fungi. The ability of LiP activity production by C. gallica, C. unicolor, F. trogii, T. ochracea, and T. zonatus in the medium containing mandarin-peels was reported for the first time. Several factors, such as supplementation of the nutrient medium with a variety of lignocellulosic materials, nitrogen source or surfactant (Tween 80, Triton X-100) significantly influenced production of LME by a novel strain of C. gallica. Moreover, C. gallica was found to be a promising LME producer with a potential for an easy scale up cultivation in a bioreactor and high enzyme yields (Lac-9.4 U/mL, MnP-0.31 U/mL, LiP-0.45 U/mL).

Enzymatic mechanisms and detoxification of dry olive-mill residue by Cyclocybe aegerita, Mycetinis alliaceus and Chondrostereum purpureum

The detoxification of dry olive-mill residue (DOR), a lignocellulosic by-product, was investigated using three agaric fungi: Cyclocybe aegerita, Mycetinis alliaceus and Chondrostereum purpureum. The lignin-modifying enzymes (LME) secretion pattern of the above-mentioned fungi such as DyP-type peroxidase (DyP), laccase (Lac), unspecific peroxygenase (UPO), and manganese peroxidase (MnP) was determined in presence and absence of DOR. In C. aegerita, a laccase (Lac) and unspecific peroxygenase (UPO) induction was found when DOR was present in the medium. Thus, these enzymes appeared to be responsible for DOR detoxification and, indirectly, its plant-growth-promoting effect. In the experiment performed with M. alliaceus, no differences were found in DyP-type peroxidase (DyP) secretion when the basal barley medium was supplemented with DOR. However, MnP and Lac activities in DOR-barley reached a maximum after 5 weeks of incubation with a concomitant decline in DOR phytotoxicity. C. purpureum completely eliminated DOR phytotoxicity but no significant production of LME was detected in soy or barley fungal media. Other enzymatic mechanisms were also investigated, in relation to hydrolytic enzymes and the intracellular system cytochrome P450 monooxygenase (CYPs). Our results suggest the participation of a complex enzymatic system (intra and extracellular) in C. purpureum for the biotransformation of DOR.

Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens, Phlebia radiata

As a discarded lignocellulosic biomass, chestnut shell is of great potential economic value, thus a sustainable strategy is needed and valuable for utilization of this resource. Herein, the feasibility of biological processes of chestnut shell with Dichomitus squalens, Phlebia radiata and their co-cultivation for lignin-modifying enzymes (LMEs) production and biodegradation of this lignocellulosic biomass was investigated under submerged cultivation. The treatment with D. squalens alone at 12days gained the highest laccase activity (9.42±0.73Umg(-1)). Combined with the data of laccase and manganese peroxidase, oxalate and H2O2 were found to participate in chestnut shell degradation, accompanied by a rapid consumption of reducing sugar. Furthermore, specific surface area of chestnut shell was increased by 77.6-114.1% with the selected fungi, and total pore volume was improved by 90.2% with D. squalens. Meanwhile, the surface morphology was observably modified by this fungus. Overall, D. squalens was considered as a suitable fungus for degradation of chestnut shell and laccase production. The presence of LMEs, H2O2 and oxalate provided more understanding for decomposition of chestnut shell by the white-rot fungi.

Effect of copper, nutrient nitrogen, and wood-supplement on the production of lignin-modifying enzymes by the white-rot fungus Phlebia radiata

Production of the oxidoreductive lignin-modifying enzymes – lignin and manganese peroxidases (MnPs), and laccase – of the white-rot basidiomycete Phlebia radiata was investigated in semi-solid cultures supplemented with milled grey alder or Norway spruce and charcoal. Concentrations of nutrient nitrogen and Cu-supplement varied also in the cultures. According to extracellular activities, production of both lignin peroxidase (LiP) and MnP was significantly promoted with wood as carbon source, with milled alder (MA) and low nitrogen (LN) resulting with the maximal LiP activities (550 nkat l−1) and noticeable levels of MnP (3 μkat l−1). Activities of LiP and MnP were also elevated on high nitrogen (HN) complex medium when supplemented with spruce and charcoal. Maximal laccase activities (22 and 29 μkat l−1) were obtained in extra high nitrogen (eHN) containing defined and complex media supplemented with 1.5 mM Cu2+. However, the nitrogen source, either peptone or ammonium nitrate and asparagine, caused no stimulation on laccase production without Cu-supplement. This is also the first report to demonstrate a new, on high Cu2+ amended medium produced extracellular laccase of P. radiata with pI value of 4.9, thereby complementing our previous findings on gene expression, and cloning of a second laccase of this fungus.

The synergistic effect on production of lignin-modifying enzymes through submerged co-cultivation of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora using agricultural residues

The lignin-modifying enzymes (LMEs) play an important role in decomposition of agricultural residues, which contain a certain amount of lignin. In this study, the production of LMEs by three co-cultivated combinations of Phlebia radiata, Dichomitus squalens and Ceriporiopsis subvermispora and the respective monocultures was comparatively investigated. Laccase and manganese peroxidases (MnP) were significantly promoted in the co-culture of P. radiata and D. squalens, and corncob was verified to be beneficial for laccase and MnP production. Moreover, laccase production by co-culture of P. radiata and D. squalens with high ratio of glucose to nitrogen was higher than low ratio under carbon- and nitrogen-meager conditions. New laccase isoenzymes measured by Native-PAGE were stimulated by co-cultured P. radiata with D. squalens or C. subvermispora, respectively, growing in the defined medium containing corncob, but the expression of laccase was greatly restrained by the co-culturing of D. squalens with C. subvermispora. This study showed that the synergistic and depressing effects of co-cultivation of P. radiata, D. squalens and C. subvermispora on LMEs were species specific.

Co-cultured Production of Lignin-Modifying Enzymes with White-Rot Fungi

Co-cultivation was a potential strategy in lignocellulolytic biodegradation with producing high activity enzymes due to their synergistic action. The objective of this study was to investigate the rarely understood effects of co-culturing of two white-rot fungi on lignin-modifying enzymes (LMEs) production. Six species, Bjerkandera adusta, Phlebia radiata, Pleurotus ostreatus, Dichomitus squalens, Hypoxylon fragiforme and Pleurotus eryngii, were cultured in pairs to study the production of LMEs. The paired hyphal interaction observed showed that P. eryngii is not suitable for co-growth. The use of agar plates containing dye RBBR showed elevated decolourisation at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. radiata with D. squalens under submerged cultivation; the highest value was measured after 4 days of incubation (120 U mg(-1)). The improved productions of MnP and LiP were simultaneously observed at the co-culture of P. ostreatus and P. radiata (MnP = 800 nkat L(-1) after 4 days of incubation; LiP = 60 nkat L(-1) after 7 days of incubation), though it was not a good producer of laccase. P. ostreatus appeared to possess specific potential to be used in co-cultured production of LMEs. The phenotype of LMEs production was not only dependent on the species used but also regulated by different nutritions available in the culture medium. The present data will provide evidence for illustrating the regulatory roles of C/N on LMEs production under the co-cultures' circumstances.

A new strain of Bjerkandera sp. production, purification and characterization of versatile peroxidase

The lignin modifying enzymes (LMEs) secreted by a new white rot fungus isolated from Chile were studied in this work. This fungus has been identified as a new anamorph of Bjerkandera sp. based on the sequences of the ribosomal DNA and morphological analysis at light microscopy showing hyaline hyphae without clamp connection, cylindrical conidia and lack of sexual forms, similar to those reported in other Bjerkandera anamorphs. The characterization of the culture medium for the highest LMEs production was performed in flask cultures, with a formulation of the culture medium containing high levels of glucose and peptone. The highest Mn-oxidizing peroxidase activity (1,400 U/L) was achieved on day 6 in Erlenmeyer flasks. Four peroxidases (named R1B1, R1B2, R1B3 and R1B4), have been purified by using ion-exchange and exclusion molar chromatographies. All of them showed typical activity on Mn2+ and exhibited Mn-independent activity against 2,6-dimethoxyphenol. R1B4 showed also activity on veratryl alcohol (pH 3) indicating that this enzyme belongs to the versatile peroxidase family. The high VP production capacities of this strain, as well as the enzymatic characteristics of the LMEs suggest that it may be successfully used in the degradation of recalcitrant compounds.

Controversial role of fungal laccases in decreasing the antibacterial effect of olive mill waste-waters

Antibacterial diffusion tests (against Bacillus megaterium) detected both bacterial growth-promoting and growth-inhibiting components in olive mill waste-water (OMW). Mixtures of OMW aromatic components showed antibacterial effects that did not show antibacterial activity when tested as individual compounds. Strains of white rot fungi (WRF) producing different patterns of lignin modifying enzymes (LMEs) have been evaluated for OMW remediation under nutritional conditions affecting the LMEs produced. The decrease of both the content in OMW phenols and in the OMW antibacterial activity was compared with fungal growth and LMEs production. OMW addition to the cultures increased fungal growth and laccase activity irrespectively of the nutritional conditions of the cultures. Laccase was the sole LME activity that increased after OMW addition to the cultures. Neither the increased growth of WRF in OMW-containing cultures, their content in laccase nor the amount of OMW phenols were direct indications of a greater decrease in OMW antibacterial effect. The higher decrease in OMW antibacterial activity was obtained in cultures of Phanerochaete flavido-alba in an N-rich media.

Production of lignin modifying enzymes by co-cultivated White-rot fungi Cerrena maxima and Coriolus hirsutus and characterization of laccase from Cerrena maxima

The lignin modifying enzymes production by co-cultivated C. hirsutus and Cerrena maxima, pure fungal cultures Coriolus hirsutus and Cerrena maxima in defined medium under static and shaken conditions, in media with birch sawdust and sulfonate lignin and under SSF of birch sawdust was comparatively studied. Analysis of the structure of residual lignin before and after fungal treatment indicated that the oxidation of phenolic component of lignin occurred and the residual lignin is enriched in carboxylic acid groups. Three laccase isoenzymes are produced by Cerrena maxima growing in defined media. The isoenzyme with pI 3.5 was largely present and its molecular mass, isoelectric point, metal content and carbohydrate composition were determined. The laccase was characterized to have a broad substrate specificity. It belongs to high redox potential laccases (750 mV vs. normal hydrogen electrode) and might be useful in industrial applications.

Effect of different carbon and nitrogen regimes on Poly R decolorization by white-rot fungi

Production of lignin-modifying enzymes by 10 white-rot fungi, as measured by decolorization of Poly R 478 dye, varied in response to different carbon and nitrogen regimes. Fastest decolorization rates were achieved with wood polysaccharide monomers (glucose, xylose) as carbon source, although cellulose was the only carbon source to facilitate decolorization by all test fungi. Enzyme production by most isolates was strongly dependant on nitrogen levels, with high nitrogen conditions generally suppressing enzyme production. One unidentified isolate (HKUCC 4062) displayed apparent nitrogen deregulation of enzyme production. A cellulose-low nitrogen growth medium is recommended for agar-based screening procedures of lignin-modifying enzyme production by white-rot fungi.

Decolorization of molasses spent wash by the white-rot fungus Flavodon flavus, isolated from a marine habitat.

Flavodon flavus (Klotzsch) Ryvarden, a basidiomycete (NIOCC strain 312) isolated from decomposing leaves of a sea grass, decolorized pigments in molasses spent wash (MSW) by 80% after 8 days of incubation, when used at concentrations of 10% and 50%. Decolorizing activity was also present in media prepared with half-strength seawater (equivalent to 15 ppt salinity). Decolorizing activity was seen in low-nitrogen medium, nutrient-rich medium and in sugarcane bagasse medium. The percentage decolorization of MSW was highest when glucose or sucrose was used as the carbon source in the low-nitrogen medium. The production of lignin-modifying enzymes, manganese-dependent peroxidase (MNP) and laccase decreased in a medium containing MSW. MNP production and MSW decolorization were inversely correlated, suggesting no role for MNP in MSW decolorization. The decolorization of MSW was not effective when F. flavus was immobilized in calcium alginate beads. Decolorization was achieved best in oxygenated cultures. Besides color, total phenolics and chemical oxygen demand were reduced by 50% in MSW treated with F. flavus, suggesting its potential in the bioremediation of effluents.

Lignin-modifying enzymes of flavodon flavus, a basidiomycete isolated from a coastal marine environment

A basidiomycetous fungus Flavodon flavus (Klotzsch) Ryvarden (strain 312), isolated from decaying sea grass from a coral lagoon off the west coast of India, mineralized nearly 24% of 14C-labeled synthetic lignin to 14CO2 in 24 days. When grown in low-nitrogen medium (2.4 mM N) this fungus produced three major classes of extracellular lignin-modifying enzymes (LMEs): manganese-dependent peroxidase (MNP), lignin peroxidase (LIP), and laccase. Low MNP and laccase activities were seen in high-nitrogen medium (24 mM N), but no LIP activity was seen. In media containing lignocellulosic substrates such as pine, poplar, or sugarcane bagasse as the sole source of carbon and nitrogen, relatively high MNP and moderate levels of laccases were seen, but LIP production either was not seen or was minimal. LME production was also seen in media prepared with artificial seawater. Fast protein liquid chromatography and isoelectric focusing resolved LMEs into four isozymes each of MNP and LIP, while laccase isozymes were resolved into two groups, one group containing seven isozymes (pIs 4 to 6) and the other group containing at least three isozymes (pIs < 3). The molecular masses of the different isozymes were 43 to 99 kDa for MNP, 40 and 41.5 kDa for LIP, and 43 and 99 kDa for laccase. F. flavus showed effective degradation of various dye pollutants in media prepared with or without artificial seawater. This is the first report on the production of all three major classes of LMEs by F. flavus and points to the bioremediation potential of this organism in terrestrial as well as marine environments.