Fungus Pleurotus Pulmonarius Research Articles

Purification and characterization of laccase from the white-rot fungus Pleurotus pulmonarius VAST02.42

Purification and characterization of laccase from the white-rot fungus Pleurotus pulmonarius VAST02.42

Degradation of zearalenone and aflatoxin B1 by Lac2 from Pleurotus pulmonarius in the presence of mediators

The contamination of foods and feeds with mycotoxins has been an issue of global significance. For mycotoxin detoxification, enzymatic biodegradation using laccase has received much attention. In this study, a laccase gene lac2 from the fungus Pleurotus pulmonarius was expressed in the Pichia pastoris X33 yeast strain to produce recombinant proteins. Enzymatic properties of recombinant Lac2 and its ability to degrade zearalenone (ZEN) and Aflatoxin B1 (AFB1) in the presence of four mediators (ABTS, TEMPO, AS and SA) were investigated. Result showed that the optimum pH and temperature of recombinant Lac2 were 3.5 and 55 °C, respectively. Lac2 was not sensitive to heat and stable under both acidic and alkaline conditions. Lac2-ABTS and Lac2-AS were efficient systems for ZEN degradation over a wide range of pH (4–8) and temperature (40–60 °C). Lac2-AS was the most efficient system for AFB1 degradation, reaching 99.82% of degradation at pH 7 and 37 °C after 1 h of incubation. Finally, the Lac2-mediator oxidation products were structurally characterized. This study lays a solid foundation for the application of Lac2 laccase combined with AS for degrading mycotoxin in food and feed.

White rot fungus Pleurotus pulmonarius enhanced bioremediation of highly PCDD/F-contaminated field soil via solid state fermentation

This study was performed to evaluate the use of white rot fungus, Pleurotus pulmonarius, to treat polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) in contaminated soil using solid state fermentation (SSF). The soil was collected from a long-closed pentachlorophenol plant in southern Taiwan. The non-sterilized soil with a total PCDD/F concentration of 14,000 ± 2400 ng I-TEQ kg−1 was mixed directly with the solid fungal inocula at dry w/w ratio of 1:1.4 (ratio-adjusted test) and incubated at 26 ± 2 °C in a controlled environment. The highest PCDD/F decomposition was observed during the mycelium colonization. Pearson correlation coefficient (r) studied during this period (35 days) indicated that laccase had no significant correlation (r = −0.53), while manganese peroxidase had a strong positive correlation (r = 0.88) with PCDD/F decomposition efficiency. After 72 days, the more toxic congeners, tetra- and penta-CDD/Fs were removed to non-detectable levels. Meanwhile, the removal efficiencies of hexa-, hepta-, and octa-CDD/Fs were >80%, >97%, and >90%, respectively. The simultaneous degradation of low and high chlorinated DD/Fs suggested that overall removal was nonspecific. The overall PCDD/F removal was 96%, and the residual concentration (276 ng I-TEQ kg−1) was below the regulatory control limit (1000 ng I-TEQ kg−1). In conclusion, this study shows that P. pulmonarius via SSF can successfully remediate the PCDD/F-contaminated field soil. Furthermore, this SSF technique overcame the well-known intractability of PCDD/F biodegradation in non-sterilized soil, making it promising for actual field application.

The plant growth-promoting potential of the mesophilic wood-rot mushroom Pleurotus pulmonarius.

To demonstrate the plant growth-promoting potential of a wood-decay mushroom. A wild strain of a white rot fungus (Pleurotus pulmonarius) was found to convert 10mmoll-1 L-tryptophan (TRP) to approximately 15μgml-1 indole-3-acetic acid (IAA) under the optimal growth conditions of 30°C and pH 5 for 15days. Results of gas chromatography-mass spectrometry indicated IAA synthesis through the indole-3-pyruvic acid pathway when using cellulose as a sole carbon source. The mycelium as well as the culture filtrate promoted the growth and chlorophyll content of seedlings. In a monocotyledonous plant (rice), the number of lateral roots was increased experimentally, whereas in a dicotyledonous plant (tomato), the fungus led to an increased length of shoots and roots. TRP-dependent IAA production was demonstrated for the first time for P. pulmonarius and may be responsible for enhancing plant growth in vitro. Synthesis of IAA as the most prevalent phytohormone in plants has been demonstrated for soil microfungi. Pleurotus pulmonarius is reported as an IAA-producing wood-decay macrofungus. The higher temperature optimum of P. pulmonarius isolated from subtropical environment compared to other Pleurotus species from temperate regions makes it more suitable for application in subtropical/tropical regions.

Comparative qualitative profile of various extracellular enzymes produced by two indigenous fungus Lentinus cladopus and Pleurotus pulmonarius

Comparative qualitative profile of various extracellular enzymes produced by two indigenous fungus Lentinus cladopus and Pleurotus pulmonarius

Effect of basidiomycete fungi on the discoloration and phytotoxicity of synthetic dye and textile effluent

The elimination of toxic wastes from industrial activities, mainly the textile industry, has induced the researchers to seek new techniques that reduce or eliminate the toxicity of these effluents. The textile effluent has a high chemical demand of oxygen and strong coloration, requiring an especific treatment. The aim of this study was to evaluated the decolorizationRemazol Brilliant Blue R (RBBR) and textile effluent using pre-selected cultures of basidiomycete fungi: Lentinula edodes, Pleurotusostreatus and Pleutotuspulmorarius, and phytotoxicity of the dye and effluent before and after treatment with fungi. The decolorization test was realized in a liquid medium and the absorbance determined in spectrophotometer. For the dye was used to two pH values (5.0 and 9.0) and concentration (0.1 gL-1 and 0.5 gL-1). Lactuca sativa L. seeds were exposed to dye samples and textile effluents and the parameters evaluated were the germination rate and root lenght. The fungus Pleurotuspulmonarius was the one with the best result on the decolorization of dye RBBR on the both values: pH and concentration. As the textile effluent there was no significant difference among the treatments. In some treatments with the dye germination rate decreased indicating toxicity after decolorization. However there was an increase in root growth in the presence of the dye treated with P.pulmonarius.

White Rot Fungus (Pleurotus pulmonarius) Cultivated on Lead Contaminated Rice Straw Induced Haematotoxicity and Lead Accumulation in Liver and Kidney of Wistar Rats

White rot fungi (Pleurotus species) are edible mushrooms known for their ability to bioaccumulate contaminants including metals from the environment. There is scarcity of information on the toxic effects of the bioaccumulated metals on organisms at higher trophic levels. In this study, we investigated alterations in haematological indices and erythrocyte morphology and lead bioaccumulation potentials in liver and kidney of Wistar rats fed aqueous extract of Pleurotus pulmonarius cultivated on lead (0, 10, 100, 200, 500 and 1000 mg/L) contaminated rice straw substrate. After 8 weeks of cultivation, mushroom did not germinate in the 1000 mg/L Pb contaminated rice straw. 1 g of mushroom harvested from each of the remaining concentrations showed significant (p<0.001) increase in Pb concentration. Rats exposed to 0.5 mL of the contaminated mushroom extracts for 30 days, showed decreased leucocyte, erythrocyte, haematocrit, platelet and haemoglobin concentrations. Abnormal erythrocyte morphologies like acanthocytes, schizocytes and tear drop were significantly higher in the Pb treated mushroom fed rats than the control. The kidney and liver of treated rats showed significant (p<0.05) increase in Pb concentration with higher Pb bioaccumulation factor in the kidney than the liver. Also insignificant decrease in body and organ weight was observed in treated rats than the control. Pb accumulation in P. pulmonarius increased liver and kidney Pb bioaccumulation and induce alterations in haematological indices and erythrocyte morphology in rats. This poses public health threat to humans and other tertiary consumers foraging white rot fungi from metal contaminated sites.

Potential of the White-Rot Fungus Pleurotus pulmonarius F043 for Degradation and Transformation of Fluoranthene

Fluoranthene, a four-ring polycyclic aromatic hydrocarbon that is possible genotoxic in nature, has been used as an indicator for assessing polycyclic aromatic hydrocarbon (PAH)-containing pollutants. Microbial degradation is one of the promising methods in removing up PAH-contaminated environments. White-rot fungi have showed the ability to degrade a wide range of PAHs. This study aimed to investigate enzyme production, fungal biomass, and glucose utilization during the biodegradation process of fluoranthene by a white-rot fungus Pleurotus pulmonarius F043 and to identify the metabolites produced in the degradation process. The extracellular ligninolytic enzyme system of the fungi, producing laccases and peroxidases, was directly linked to the biodegradation of fluoranthene. The production of ligninolytic enzymes during fluoranthene degradation was related to an increase in the biomass of Pleurotus pulmonarius F043. Fluoranthene removal decreased with an increase in fluoranthene concentrations. The highest biomass production of Pleurotus pulmonarius F043 (≥ 4 400 mg L—1) was found in the 10 mg L—1 fluoranthene culture after 30 d of incubation. Two fluoranthene metabolites, naphthalene-1,8-dicarboxylic acid and phthalic acid, were found in the process of fluoranthene degradation. Laccase was revealed as the major enzyme that played an important role in degradation process. Suitable conditions must be found to promote a successful fungal biotransformation augmentation in liquid culture.

Optimization of pyrene degradation by white-rot fungus Pleurotus pulmonarius F043 and characterization of its metabolites

Pleurotus pulmonarius F043, a fungus collected from tropical rain forest, was used to degrade pyrene, a four-rings polycyclic aromatic hydrocarbons (PAHs), in a mineral medium broth. A maximum degradation rate of pyrene (90 %) was occurred at pH 3 and the lowest degradation rate was found in the culture at pH 10 (2 %). More than 90 % pyrene degradation was achieved at pH ranged from 3 to 5, whereas the degradation rate significantly declined when the pH was >5. The degradation of pyrene increased from 2 to 96 % when the temperature rose from 4 to 25 °C. When the temperature was increased to 60 °C resulting the lowest degradation rate into 7 %. Among the agitation rates tested, 120 rpm was the best with 95 % degradation, followed by 100 rpm (90 %). The optimum agitation range for pyrene degradation by P. pulmonarius F043 was 100-120 rpm. Among all the concentrations tested, 0.5 % Tween 80 was the best with 98 % degradation, followed by 1 % Tween 80 (90 %). The optimum concentration of Tween 80 for pyrene degradation by P. pulmonarius F043 was 0.5-1 %. The degradation rate decreased, while the concentration of Tween 80 was increased. The metabolic product was found during degradation process through the identification of gentisic acid by TLC, UV-Spectrophotometer, and GC-MS.

Production of laccase and manganese peroxidase by Pleurotus pulmonarius in solid-state cultures and application in dye decolorization

The production of ligninolytic enzymes (laccase and Mn-dependent peroxidase) by the white-rot fungus Pleurotus pulmonarius (FR.) Quélet was studied in solid-state cultures using agricultural and food wastes as substrate. The highest activities of laccase were found in wheat bran (2,860 ± 250U/L), pineapple peel (2,450 ± 230U/L), and orange bagasse (2,100 ± 270U/L) cultures, all of them at an initial moisture level of 85%. The highest activities of Mn peroxidase were obtained in pineapple peel cultures (2,200 ± 205U/L) at an initial moisture level of 75%. In general, the condition of high initial moisture level (80-90%) was the best condition for laccase activity, while the best condition for Mn peroxidase activity was cultivation at low initial moisture (50-70%). Cultures containing high Mn peroxidase activities were more efficient in the decolorization of the industrial dyes remazol brilliant blue R (RBBR), Congo red, methylene blue, and ethyl violet than those containing high laccase activity. Also, crude enzymatic extracts with high Mn peroxidase activity were more efficient in the in vitro decolorization of methylene blue, ethyl violet, and Congo red. The dye RBBR was efficiently decolorized by both crude extracts, rich in Mn peroxidase activity or rich in laccase activity.

Copper improves the production of laccase by the white-rot fungus Pleurotus pulmonarius in solid state fermentation

Pleurotus pulmonarius (Fr) Quélet, produced laccase as the main ligninolytic enzyme when cultivated on solid-state cultures using corn cob as substrate. The addition of copper greatly increased the production of enzyme. The addition of 25.0 mM CuSO4 increased the level of laccase from 270 to 1,420 U.L-1 and the fungus showed high resistance to copper under the conditions used in this work.

Toxicities of DDE on Wheat and Bioremediation of DDE by Fungus Pleurotus pulmonarius

ABSTRACT An in vitro study was performed to determine the acute phytotoxicities and genotoxicity of DDE either spiked to soil or added to hydrophonic cultures on wheat Triticum aestivum. A 24-well plate was first used to determine toxicity on individual grains using conventional seed germination/seedling growth toxicity tests whereas a single cell electrophoresis system was applied to measure genotoxicity at single cell level for wheat. Hydrophonic cultures provide a simplified environment to screen for toxicities with high sensitivity. Inverse dose-response relationships were detected between exogenous DDE levels and one of the following parameters: seed germination, seedling growth, and genotoxicity. In contrast, soil reduced the stress on T. aestivum by lowering bioavailability leading to less DDE distributed in radicle and coleoptile, modulated growth, and enhanced tolerance. At all DDE doses spiked to soil including the reference safety level of 0.5 mg/kg, DNA breakage was detected in both radicle and coleoptile but their magnitudes did not correlate with the organ nor the soil DDE contents. Thus, although wheat is highly sensitive to the genotoxic effect of DDE, first demonstrated here, the seed germination test offers a simple quantitative measure of DDE's phytotoxicity in soil and hydrophonic cultures. This study also found that fungus Pleurotus pulmonarius, which secretes extracellular ligninolytic enzymes causing non-specific cleavage of lignin and organopollutants, remediated DDE spiked to soil. In 5 weeks, 78% of 10 mg/kg DDE was biodegraded, and the fungal-treated soil reduced acute toxicity on T. aestivum using the seed germination test.

Co-production of ligninolytic enzymes byPleurotus pulmonarius on wheat bran solid state cultures

In this paper, the production of biomass and ligninolytic enzymes by the white-rot fungus Pleurotus pulmonarius (Fr.) Quélet cultured on wheat bran at high initial moisture was evaluated. When the initial moisture was lower than 86%, the fungal hyphae penetrated into and bound tightly to the solid-substrate particles. When the moisture was equal or higher than 86%, the growth was characterized by formation of a large mycelial mass above the substrate (surface growth). In this case, the mycelial mass could be easily separated from the residual solid substrate and quantified by gravimetric analysis. The fungus produced 0.32 g of dry mycelial mass per g of dry substrate after 15 days of cultivation. Analysis of the residual substrate showed that growth was mainly due to the consumption of soluble proteins and carbohydrates. The condition of high initial moisture strongly promoted the expression of laccase (up to 24,000 U per g of substrate or 78,000 U per g of dry fungal biomass), while the production of manganese peroxidase was negatively affected. In fact, manganese peroxidase was maximally produced when the initial moisture was 75% (2,000 U per g of substrate). The production of other enzymes, such as polysaccharidases and proteases, was not significantly affected by the initial moisture.

Purification and characterization of the main laccase produced by the white-rot fungus Pleurotus pulmonarius on wheat bran solid state medium.

The wood-degrading fungus Pleurotus pulmonarius produces at least two laccase isoforms, Lcc1 and Lcc2, when grown on wheat bran solid state medium. The main laccase, Lcc2, was purified to apparent electrophoretic homogeneity by using acetone precipitation, anion-exchange chromatography and gel filtration. Lcc2 had been purified 5.9-fold with a yield of 49%. A specific activity of 19750 U/mg protein was found using syringaldazine as a substrate under standard assay conditions. The enzyme is a homodimeric glycoprotein containing 44% glycosilation and an apparent molecular mass of 46 kDa. Type I and type III Cu(2+) centers were identified by spectrophotometry. The laccase showed optimal activity at pH 6.2-6.5, 4.0-5.5, and 6.0-8.0 with syringaldazine, ABTS and guaiacol as substrates, respectively. For all substrates, the highest oxidation rates were obtained at 50 degrees C. The enzyme was stable over a large range of pH (4.5-8.0) and at temperatures up to 50 degrees C. Under standard assay conditions, the apparent K(M) values were 12, 210 and 550 microM for syringaldazine, ABTS and guaiacol, respectively. Purified Lcc2 was strongly inhibited by sodium azide, 2-mercaptoethanol and Hg(2+), and slightly inhibited by Mn(+2) and the chelant agents, EDTA and EGTA. The enzyme was activated by Cu(2+) and it retained a high percentage of its activity in the presence of organic solvents, such as acetonitrile and acetone.

Decolourisation of industrial dyes by solid-state cultures of Pleurotus pulmonarius

A solid-state system to obtain rapid in vivo industrial dye decolourisation by the white-rot fungus Pleurotus pulmonarius is proposed. When cultivated on glucose\\ammonium tartrate–corncob solid-state medium, P. pulmonarius decolourised structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes). Amido black, congo red, trypan blue, methyl green, remazol brilliant blue R (RBB), methyl violet, ethyl violet and brilliant cresyl blue were completely decolourised after 6 days of cultivation, while methylene blue and Poly R-478 were partially decolourised. The capability of fungal culture to decolourise industrial dyes appears to be due to high titres of laccase (480 U/ml) produced in response to the presence of soluble phenolics.

Purification and Characterization of a Benzo[a]pyrene Hydroxylase from Pleurotus pulmonarius

Cytochrome P450 has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by white-rot fungi. We have purified and reconstituted a benzo[a]pyrene hydroxylating cytochrome P450 (P450) from microsomal fractions of the white rot fungus Pleurotus pulmonarius. The microsomal P450 was recovered using a combination of n-aminooctyl agarose and hydroxyapatite chromatography and had an apparent molecular mass of 55 kDa. The purified protein exhibited moderate affinity for benzo[a]pyrene with a Ks of 66 μM calculated from the Type I substrate binding spectra produced. Reconstitution of activity was achieved and a turnover of 0.75 nmol 3-hydroxybenzo[a]pyrene product/min/nmol P450 was observed, comparable to levels of metabolism observed by animal cytochromes P450 involved in xenobiotic detoxification.

Induction of linoleic acid-supported benzo(a)pyrene hydroxylase activity by manganese in the white rot fungus Pleurotus pulmonarius

Linoleic acid (LA)-supported benzo(a)pyrene (BAP) hydroxylation was observed in the cytosolic fraction of the white rot fungus Pleurotus pulmonarius after incubation of the mycelium with manganese (Mn +2). Linear relationships were obtained between the indelible activity and manganese concentration added to the fungal medium, and the LA and BAP concentration added to the enzymatic reaction mixture. Cytochrome P450 inhibitors nordihydroguaiarefc acid, ketoconazole, piperonyl butoxide and carbon monoxide inhibited the induced activity. The results suggest that the presence of Mn and LA in the fungal environment could enhance P450-mediated degradation of xenobiotic compound such as BAP.

Manganese-enhanced biotransformation of atrazine by the white rot fungus Pleurotus pulmonarius and its correlation with oxidation activity.

Manganese enhanced atrazine transformation by the fungus Pleurotus pulmonarius when added to a liquid culture medium at concentrations of up to 300 microM. Both N-dealkylated and propylhydroxylated metabolites accumulated in the culture medium, with the former accumulating to a greater extent than did the latter. Lipid peroxidation, oxygenase and peroxidase activities, and the cytochrome P-450 concentration increased. In addition, an increase in the spectral interactions between atrazine and components in the cell extract was observed. Antioxidants, mainly nordihydroguaiaretic acid, which inhibits lipoxygenase, peroxidase, and P-450 activities, and piperonyl butoxide, which inhibits P-450 activity, inhibited atrazine transformation by the mycelium. It is suggested that the stimulation of oxidative activity by Mn might be responsible for increasing the biotransformation of atrazine and for nonspecific transformations of other xenobiotic compounds.

Degradation of atrazine by the lignocellulolytic fungus Pleurotus pulmonarius during solid-state fermentation

The biotransformation of atrazine added to a mixture of cotton and wheat straw (CWS) and inoculated with the white-rot fungus, Pleurotus pulmonarius, was studied, as a proposed system for bioremediation. The concentration of methanol-extractable atrazine was reduced, due to both biological transformation and physical-chemical adsorption to the straw. Only 32% of the total radioactivity added as 14C-ring-labeled atrazine to pasteurized CWS inoculated with Pleurotus was extracted two weeks after fungal colonization, and less than 70% from non-inoculated CWS. The reduction in extractable radioactivity increased with time of incubation. No mineralization of the triazine ring was found during six weeks of incubation, but transformation to two groups of atrazine metabolites, chlorinated and dechlorinated, occurred, as a result of the activity of the fungus inoculated and natural bacterial population. Unextractable radioactivity was recovered after digesting the colonized substrate with H 2SO 4, indicating adsorption of the herbicide and its metabolites to the straw. The results suggest that this process can be used to detoxify atrazine by both adsorption and biodegradation.

Effect of medium composition on 1-octen-3-ol formation in submerged cultures of Pleurotus pulmonarius

The effect of nitrogen and fatty-acid-rich substrates on the production of 1-octen-3-ol by the edible fungus Pleurotus pulmonarius, during growth in both shaken flask and fermentor cultures, and in-vitro, in post-harvested mycelium, was studied. Addition of soybean flour and soybean oil to the growth medium enhanced 1-octen-3-ol production about sevenfold and doubled the fungal biomass, as compared to that obtained from P. pulmonarius cultured on a defined synthetic medium. A clear relationship between the production of 1-octen-3-ol and lipoxygenase activity was found during the growth of mushroom pellets. The highest in-vitro generation of 1-octen-3-ol was obtained upon addition of exogenous linoleic acid and pure O2 to pellets grown with soybean fluor and soybean oil. This generation was even higher than that of fruiting bodies exposed to the same conditions. These results suggest that lipoxygenase activity and, subsequently, 1-octen-3-ol biosynthesis in P. pulmonarius are enhanced by the presence of substrates containing fatty acids in the growth medium.