Enzymatic decolorization of melanin by lignin peroxidase from Phanerochaete chrysosporium.

Skin darkening results as a consequence of the accumulation of skin pigment melanin. To combat this, the amplitude of skin lightening agents are commercially available, most of which inhibit melanin synthesis. Decolorization of melanin is an alternative method of skin lightening. In this study, we show that lignin peroxidase (LiP), an extracellular enzyme purified from Phanerochaete chrysosporium NK-1 isolated from a forest soil can effectively degrade and decolorize melanin in vitro. Decolorization conditions including pH, temperature, incubation time, enzyme concentration, and mediator addition were investigated to optimize the reaction conditions. The results indicate that pH 3, 40 °C, 15 IU/ml, and 10 h incubation were the optimal conditions for the decolorization of the melanin. The use of the mediator, veratryl alcohol was also found effective to enhance the efficacy of the melanin decolonization, with up to 92% decolorization. The scanning electron microscopy results showed void spaces on the treated melanin granules as compared to the untreated sample, indicating the degradation of melanin. Changes in the fingerprint region of the melanin were observed. Between wavenumbers 1500-500 cm-1, for example, the presence of new peaks in the treated melanin at 1513, 1464, and 1139 cm-1 CH2, CH3 bend and C-O-C stretch represented structural changes. A new peak at 2144 cm-1 (alkynyl C≡C stretch) was also detected in the decolorized melanin. The cytotoxicity study has shown that the treated melanin and LiP have low cytotoxic effects; however, the mediator of veratryl alcohol could result in high mortality which suggests that its use should be meticulously tested in formulating health and skincare products. The findings of the study suggest that LiP produced by Phanerochaete chrysosporium has the potential to be used in the medical and cosmetic industries, particularly for the development of biobased cosmetic whitening agents.

Self-fusion and fusion cell isolation of transformants derived from white rot fungus Phanerochaete sordida YK-624 by simple visual method.

In order to develop a simple method for crossing two transformants, we first attempted to elucidate the fusion type (self-compatibility or -incompatible) of Phanerochaete sordida YK-624. Two transformants expressing green or red fluorescent protein derived from an auxotrophic mutant were constructed. Each recombinant protein fluoresced by expression as a fused protein with glyceraldehyde-3-phosphate dehydrogenase. On co-culture of both transformants, a number of sequential hyphal cells emitting dual fluorescence were formed at the contact areas of both hyphae. Some of the single cells isolated as protoplasts and chlamydospore from the co-cultures also expressed these fluorescent proteins. These results suggest that P. sordida YK-624 possesses a self-compatible fusion system. In addition, transformant strains with different fluorescence derived from this fungus can readily undergo self-fusion and nuclear interchange events by confrontational and mixed cultivation, and we developed a simple method that allows fused cells to be isolated as chlamydospores.

Enzymatic Textile Dyes Decolorization by In vitro and In silico Studies.

BACKGROUND: Laccase, a multicopper oxidoreductase (EC: 1.10.3.2), is a widely used enzyme in bioremediation of textile dye effluents. Fungal Laccase is preferably used as a remediating agent in the treatment and transformation of toxic organic pollutants. In this study, crude laccase from a basidiomycetes fungus, Phanerochaete sordida, was able to decolorize azo, antroquinone and indigoid dyes. In addition, interactions between dyes and enzyme were analysed using molecular docking studies. METHODS: In this work, a white rot basidiomycete's fungus, Phanerochaete sordida, was selected from forest soil isolates of Eastern Ghats, and Tirumala and lignolytic enzymes production was assayed after 7 days of incubation. The crude enzyme was checked for decolourisation of various synthetic textile dyes (Vat Brown, Acid Blue, Indigo, Reactive Blue and Reactive Black). Molecular docking studies were done using Autodock-4.2 to understand the interactions between dyes and enzymes. RESULTS: Highest decolourisation efficiency was achieved with the crude enzyme in case of vat brown whereas the lowest decolourisation efficiency was achieved in Reactive blue decolourisation. Similar results were observed in their binding affinity with lignin peroxidase of Phanerochaete chrysosporium through molecular docking approach. CONCLUSION: Thus, experimental results and subsequent in silico validation involving an advanced remediation approach would be useful to reduce time and cost in other similar experiments.

Phanerochaete sordida as a cause of pulmonary nodule in an immunocompromised patient: a case report.

BACKGROUND: Phanerochaete sordida is a species of wood rotting fungus, which can degrade lignin, cellulose and hemicellulose contained in wood and other hard-to-biodegrade organic substances. However, to date, there have been no other reports demonstrating that P. sordida can infect humans. CASE PRESENTATION: A 66-year-old Japanese man presented for a mass increasing in size on his left thigh. He had been suffering from rheumatoid arthritis for 18 years and chronic obstructive pulmonary disease for 20 years, for which he was being treated with 5 mg/day prednisolone and 8 mg/week methotrexate. The mass resection was performed two months later, and was diagnosed as malignant fibrous histiocytosis. However, a computed tomography examination for tumor recurrence after surgery showed a newly emergent pulmonary nodule. We therefore decided to resect the nodule by thoracoscopic procedure. Histopathological examination of the excised specimen showed that the lesion was a granuloma, with necrotic tissue and clumping of Aspergillus-like hyphae. Therefore, the nodule was diagnosed as a fungal infection and tissue specimens were cultured microbiologically. However, fungal growth was not observed. We consequently performed genetic analysis using a broad-range polymerase chain reaction. The 28S rRNA sequence demonstrated 100% homology with P. sordida using the NCBI BLAST program against the GenBank DNA databases. CONCLUSIONS: Using broad-range polymerase chain reaction, we identified P. sordida as the causative agent of a pulmonary nodule. These findings indicate that P. sordida may be an additional opportunistic causative organism of pulmonary infection in immunocompromised patients.

Use of Swine Wastewater as Alternative Substrate for Mycelial Bioconversion of White Rot Fungi.

Seven white rot fungal species were tested for growth as mycelia using swine wastewater (SW), an agro-waste with tremendous environmental footprint, as the sole nutrient source. The SW contained high concentrations of carbon and nitrogen components, which could support nutritional requirements for mycelial growth. Out of the seven species, Pleurotus ostreatus and Hericium erinaceus were successfully cultivated on the SW medium using solid-state fermentation. Response surface methodology was employed to determine the combination of pH, temperature (T), and substrate concentration (C) that maximizes mycelial growth rate (Kr) for the two species. The optimum condition was estimated as pH = 5.8, T = 28.8 °C, and C = 11.2 g chemical oxygen demand (COD)/L for P. ostreatus to yield Kr of 11.0 mm/day, whereas the greatest Kr (3.1 mm/day) was anticipated at pH = 4.6, T = 25.5 °C, and C = 11.9 g COD/L for H. erinaceus. These Kr values were comparable to growth rates obtained using other substrates in the literature. These results demonstrate that SW can be used as an effective substrate for mycelial cultivation of the two white rot fungal species, suggesting an alternative method to manage SW with the production of potentially valuable biomass.

Bioremediation of dyes by fungi isolated from contaminated dye effluent sites for bio-usability.

Biodegradation and detoxification of dyes, Malachite green, Nigrosin and Basic fuchsin have been carried out using two fungal isolates Aspergillus niger, and Phanerochaete chrysosporium, isolated from dye effluent soil. Three methods were selected for biodegradation, viz. agar overlay and liquid media methods; stationary and shaking conditions at 25 °C. Aspergillus niger recorded maximum decolorization of the dye Basic fuchsin (81.85%) followed by Nigrosin (77.47%), Malachite green (72.77%) and dye mixture (33.08%) under shaking condition. Whereas, P. chrysosporium recorded decolorization to the maximum with the Nigrosin (90.15%) followed by Basic fuchsin (89.8%), Malachite green (83.25%) and mixture (78.4%). The selected fungal strains performed better under shaking conditions compared to stationary method; moreover the inoculation of fungus also brought the pH of the dye solutions to neutral from acidic. Seed germination bioassay study exhibited that when inoculated dye solutions were used, seed showed germination while uninoculated dyes inhibited germination even after four days of observation. Similarly, microbial growth was also inhibited by uninoculated dyes. The excellent performance of A. niger and P. chrysporium in the biodegradation of textile dyes of different chemical structures suggests and reinforces the potential of these fungi for environmental decontamination.

Bioremediation of dyes by fungi isolated from contaminated dye effluent sites for bio-usability

Biodegradation and detoxification of dyes, Malachite green, Nigrosin and Basic fuchsin have been carried out using two fungal isolates Aspergillus niger, and Phanerochaete chrysosporium, isolated from dye effluent soil. Three methods were selected for biodegradation, viz. agar overlay and liquid media methods; stationary and shaking conditions at 25 °C. Aspergillus niger recorded maximum decolorization of the dye Basic fuchsin (81.85%) followed by Nigrosin (77.47%), Malachite green (72.77%) and dye mixture (33.08%) under shaking condition. Whereas, P. chrysosporium recorded decolorization to the maximum with the Nigrosin (90.15%) followed by Basic fuchsin (89.8%), Malachite green (83.25%) and mixture (78.4%). The selected fungal strains performed better under shaking conditions compared to stationary method; moreover the inoculation of fungus also brought the pH of the dye solutions to neutral from acidic. Seed germination bioassay study exhibited that when inoculated dye solutions were used, seed showed germination while uninoculated dyes inhibited germination even after four days of observation. Similarly, microbial growth was also inhibited by uninoculated dyes. The excellent performance of A. niger and P. chrysporium in the biodegradation of textile dyes of different chemical structures suggests and reinforces the potential of these fungi for environmental decontamination.

[Breeding and characterization of laccase-producing Phanerochaete chrysosporium mutant resistant to nutritional repression].

OBJECTIVE: To screen Phanerochaete chrysosporium mutants resisting nutritional repression and to characterize laccase produced by the mutants. METHODS: We used repeated UV mutagenesis and screened the mutant strains by using the guaiacol nitrogen sufficient differential medium. We characterized enzymes production mechanism of the nutritional regulation through comparing the differences of cell growth and enzyme-production kinetics under different nutritional conditions; We validated production of laccase by Phanerochaete chrysosporium through measurements of the heat treatment, removal of manganese ion and addition of the catalase. RESULTS: Three different methods were validated that both strains of pcR5305 and pcR5324 can produce laccase under the nitrogen limitation (N-L) and nitrogen sufficient (N-S) conditions. Under the N-L conditions, pcR5305 can produce 203.5 U/L laccase and pcR5324 can produce 187.6 U/L laccase; Under the N-S conditions, pcR5305 can produce 220.6 U/L laccase and pcR5324 can produce 183.9 U/L laccase. The original strain pc530 only can produce very little laccase under either conditions. The laccase-production regulation mechanisms of the two strains are different: Production of laccase and the cell growth by pcR5305 are in synchronism. However production of the laccase by pcR5324 is repressed by nutrition. Both strains have the capacity of resisting nutritional repression and produce lignin peroxidase and manganese peroxidase with high yield. (LiP 1343.2, MnP 252.2 U/L and LiP 1169.5, MnP 172.4 U/L respectively). CONCLUSION: The mutants of Phanerochaete chrysosporium can produce laccase. At same time they showed the capacity of resisting nutritional repression and production of laccase, lignin peroxidase and manganese peroxidase. Our results possess high value for production, application and fundamental research. We provided new strains and established a very good foundation for the further research of metabolic regulation of ligninolytic enzymes production.

Desarrollo de un bioadsorbente laminar con Phanerochaete chrysosporium hipertolerante al cadmio, al níquel y al plomo para el tratamiento de aguas / Development of a laminar bioadsorbent with hypertolerant Phanerochaete chrysosporium to Cd, Ni and Pb for wastewater treatment

Antecedentes. El uso de basidiomicetes para la remoción de metales es una alternativa con respecto a los métodos tradicionales, básicamente porque la biomasa actúa como un intercambiador natural al remover metales en solución. Objetivo. Desarrollar un bioadsorbente laminar empleando el hongo basidiomicete con mayor resistencia al cadmio (Cd), al níquel (Ni) y al plomo (Pb). Métodos. Se valoró la tolerancia de Trametes versicolor, Pleurotus ostreatus y Phanerochaete chrysosporium frente a concentraciones ascendentes de sulfato de sulfato de cadmio, acetato de plomo y cloruro de níquel. Se desarrolló un bioadsorbente a base de láminas de polietileno con biomasa fúngica, que se evaluó en columnas de burbujeo empleando agua residual sintética con los 3 metales con una concentración final de 300mg/l. Finalmente, con un experimento complementario en matraces Erlenmeyer, se valoró el efecto de mayor cantidad de biomasa sobre la eficiencia de remoción. Resultados. El hongo P. chrysosporium fue la cepa más tolerante a C4H6O4Pb (10.000mg/l), a Cl2Ni (300mg/l) y CdSO4·8H2O (1.500mg/l). En el reactor y bajo condiciones no ligninolíticas, el hongo removió el 69% de la demanda química de oxígeno, produjo enzimas como lignino peroxidasa (0,01U/l) y manganeso peroxidasa (0,6U/l) y se observó la acumulación de los metales en la pared. Al incrementar la biomasa a 1,6 (p/v) se favoreció la bioadsorción de los metales en la mezcla (el 57% para el Pb, el 74% para el Cd y el 98% para el Ni) y por separado (el 95% para el Pb, el 60% para el Cd y el 56% para el Ni). Se observó una competencia entre el Ni y el Pb por los ligandos de la pared. Conclusión. Se desarrolló un novedoso sistema laminar a base de biomasa viable de P. chrysosporium que posee gran área superficial y tolera elevadas concentraciones de Cd, Ni y Pb, como alternativa para la remoción de metales en aguas(AU)

Background. The use of basidiomycetes for metal removal is an alternative to traditional methods. In this, the biomass acts as a natural ionic exchanger removing metals from solution. Objective. To develop a laminar biosorbent using a basidiomycete fungus resistant to high Cd, Ni and Pb concentrations. Methods. The tolerance of Trametes versicolor, Pleurotus ostreatus and Phanerochaete chrysosporium was evaluated using increasing concentrations of the heavy metal salts, cadmium sulphate, lead acetate and nickel chloride. A biosorbent system was developed based on polyethylene sheets with a fungal biomass. It was evaluated in bubble columns using synthetic wastewater with the 3 metal salts at a rate of 300mg/l. Finally, in a complementary experiment using shake flasks, the effect of a higher amount of biomass related to the metal removal efficiency was evaluated. Results. P. chrysosporium strain was more tolerant to C4H6O4Pb (10,000mg/l), Cl2Ni (300mg/l) and CdSO4·8H2O (1,500mg/l). In a reactor, under non-ligninolytic conditions, the fungus removed 69% of the chemical oxygen demand and produced enzymes such as LiP (0.01U/l) and MnP (0.6U/l.). An accumulation of metals in the wall was observed. By increasing the biomass to 1.6 (w/v), the metal biosorption was favored in the mixture (57% Pb, 74% Cd, and 98% Ni) and separately (95% Pb, 60% Cd, and 56% Ni). Competition between Ni and Pb by ligands of the wall was observed. Conclusion. A novel laminar system based on P. chrysosporium viable biomass was developed. It has a large surface area and tolerance to high concentrations of Cd, Ni and Pb. It seems to be an alternative for the removal of metals from water(AU)

Biodegradation of physicochemically treated polycarbonate by fungi.

Two fungal strains isolated from soil and a commercial white-rot fungus, Phanerochaete chrysosporium NCIM 1170 (SF2), were tested for biodegradation of untreated, UV-, and thermal-treated bisphenol A polycarbonate (PC). The isolated strains based on 18S rDNA analysis were characterized as Engyodontium album MTP091 (SF1) and Pencillium spp. MTP093 (SF3). About 5.4% weight loss and 40% reduction in M(n) were observed for UV-treated polycarbonate in one year with SF2 strain. An increase in surface energy and oxygen content and a reduction in methyl index indicated oxidation of PC during this period. PC exposed to the SF1 strain showed a 15 degrees C decrease in glass transition temperature, indicating an increase in the number of chain ends and, hence, an increase in the free volume of polymer. No bisphenol A, the monomer of PC, was detected during the study. NMR and FTIR spectra showed the formation of methyl groups due to pretreatments. EDAX analysis exhibited surface oxidation of the PC. The current study advocates that biodegradation of PC can be enhanced by pretreatments.

Biodegradation of endocrine-disrupting bisphenol A by white rot fungus Irpex lacteus.

Biodegradation of endocrine-disrupting bisphenol A was investigated with several white rot fungi (Irpex lacteus, Trametes versicolor, Ganoderma lucidum, Polyporellus brumalis, Pleurotus eryngii, Schizophyllum commune) isolated in Korea and two transformants of T versicolor (strains MrP 1 and MrP 13). I. lacteus degraded 99.4% of 50 mg/l bisphenol A in 3 h incubation and 100% in 12 h incubation. which was the highest degradation rate among the fungal strains tested. T. versicolor degraded 98.2% of 50 mg/l bisphenol A in 12 h incubation. Unexpectedly, the transformant of the Mn-repressed peroxidase gene of T. versicolor, strain MrP 1, degraded 76.5% of 50 mg/l bisphenol A in 12 h incubation, which was a lower degradation rate than wild-type T. versicolor. The removal of bisphenol A by I. lacteus occurred mainly by biodegradation rather than adsorption. Optimum carbon sources for biodegradation of bisphenol A by I. lacteus were glucose and starch, and optimum nitrogen sources were yeast extract and tryptone in a minimal salts medium; however, bisphenol A degradation was higher in nutrient-rich YMG medium than that in a minimal salts medium. The initial degradation of endocrine disruptors was accompanied by the activities of manganese peroxidase and laccase in the culture

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment.

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 microg-1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.

Growth substrate selection and biodegradation of PCP by New Zealand white-rot fungi.

Nine New Zealand native white-rot fungi were studied for their ability to grow and survive on different substrates formulated from bark, wheat straw, sawdust, apple pomace and maize products in order to identify their pentachlorophenol (PCP) biodegradation potential and to select a fungal carrier for bioaugmentation of polluted soils. Isolates were also evaluated to mineralize (14)C-PCP in liquid culture and in soil. The American fungus Phanerochaete chrysosporium outgrew the native fungi on the substrates tested, but the high colonisation did not result in superior PCP dechlorination as measured by chloride release. Whilst Trametes versicolor inocula produced on wheat straw and SCS (sawdust-corn meal-starch-mix) gave the highest chloride release, colonization of these two substrates as measured by biological potential was lower compared to the pomace and pomace-sawdust-mix. Neither lignin peroxidase nor manganese peroxidase production were measured for New Zealand white-rot fungi during the experiments. Laccase was the only enzyme detected. In liquid culture, the mineralisation rate was higher for T. versicolor isolates compared to P. chyrysoporium. Very little to no pentachloroanisole (PCA) was captured in the volatile fraction of T. versicolor isolates, whereas 75% of the volatile fraction of P. chrysosporium consisted of PCA. The soil microcosms studies, using contaminated soil from a timber treatment site, clearly showed that the New Zealand T. versicolor isolates mineralized PCP. Degradation of PCP in non-sterile soil was higher in the presence of white-rot fungi than in soil without white-rot fungus. This demonstrates that viable white-rot fungus is necessary for significant PCP degradation and that T. versicolor isolates showed PCP remediation potential. Wheat straw and SCS could be suitable carriers for New Zealand native T. versicolor isolates for bioremediation of PCP polluted soil sites.

Phylogenetic relationships of the genus Phanerochaete inferred from the internal transcribed spacer region.

Phanerochaete is a genus of resupinate homobasidiomycetes that are saprophytic on woody debris and logs. Morphological studies in the past indicated that Phanerochaete is a heterogeneous assemblage of species. In this study the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA was used to test the monophyly of the genus Phanerocthaete and to infer phylogenetic relationships of the 24 taxa studied. Maximum parsimony, maximum likelihood, and Bayesian analyses do not support the monophyly of the genus. However, a core group of species represented by Phanerochaete velutina, P. chrysosporium, P. sordida, P. sanguinea and others are closely related and group together in a clade. Other common Phanerochaete species including Phanerochaete rimosa, P. chrysorhiza, P. omnivora, P. avellanea, P. tiberculata, P. flava, and P. allantospora, however, do not cluster with the core Phanerochaete group.

Decolourisation of synthetic and spentwash melanoidins using the white-rot fungus Phanerochaete chrysosporium JAG-40.

Phanerochaete chrysosporium JAG-40 was isolated from the soil samples saturated with spilled molasses collected from a sugar mill. This isolate decolourised synthetic and natural melanoidins present in spentwash in liquid fermentation; up to 80% in 6 days at 30 degrees C under aerobic conditions. A large inoculum size stimulated fungal biomass production, but this gave less decolourisation of pigment; 5% w/v (dry weight) mycelial suspension was found optimum for maximum decolourisation in melanoidin medium supplemented with glucose and peptone. Gel-filtration chromatography showed that larger molecular weight fractions of melanoidin were decolourised more rapidly than small molecular weight fractions.

Degradation of primisulfuron by a combination of chemical and microbiological processes.

Microbial degradation of the herbicide primisulfuron was investigated using enrichment cultures from contaminated soils and 20 axenic cultures. At neutral pH, no disappearance of the herbicide was detected either in the enrichment cultures or in the growth media of the axenic microbial cultures. During the growth of some of the microbial strains, however, the pH of the medium dropped below 6, resulting in the hydrolysis of primisulfuron. The rate of primisulfuron hydrolysis was clearly pH dependent; primisulfuron was more persistent in neutral or weakly basic solutions than in acidic solutions. After hydrolysis of the herbicide, four products were observed. These were identified as methyl 2-(aminosulfonyl)benzoate, 2-amino-4,6-(difluoromethoxy)pyrimidine, 2-N-[[[[[4, 6-bis(difluoromethoxy)-2-pyrimidinyl]amino]carbonyl]amino]sulfonyl ]be nzoic acid, and 2-(aminosulfonyl)benzoic acid. After hydrolysis, it was found that the fungus Phanerochaete chrysosporium mineralized 27 and 24% of (14)C-phenyl- and (14)C-pyrimidine-labeled products, respectively, after 24 days of incubation. Similarly, Trametes versicolor mineralized 13 and 11% of (14)C-phenyl- and (14)C-pyrimidine-labeled hydrolysis products, respectively. In addition, primisulfuron in a hydrolytically stable solution, at pH 7. 0, was rapidly decomposed after ultraviolet irradiation, and two photolysis products were isolated [methylbenzoate and 4, 6-(difluoromethoxy)pyrimidin-2-ylurea]. When (14)C-phenyl-labeled primisulfuron was exposed to photolysis for 24 h, 32% of the initial radioactivity was recovered as (14)CO(2), whereas no (14)CO(2) was detected if the herbicide was labeled at the (14)C-pyrimidine position. Mineralization of (14)C-pyrimidine-labeled products of photolyzed primisulfuron by P. chrysosporium was approximately 25% after 24 days. These results clearly indicate that hydrolysis and photolysis of primisulfuron facilitated microbial degradation.