White Rot Fungus Pleurotus Ostreatus Research Articles

Identification and functional characterisation of ctr1, a Pleurotus ostreatus gene coding for a copper transporter

Copper homeostasis is crucial for the maintenance of life. In lignin-degrading fungi, copper is essential for the phenol oxidase enzymes that provide this activity. In this paper we report the characterization of a gene (ctr1) coding for a copper transporter in the white rot fungus Pleurotus ostreatus. The gene was identified in a cDNA library constructed from 4-day-old vegetative mycelium grown in liquid culture. The results presented here demonstrate that: (1) ctr1 functionally complements the respiratory deficiency of a yeast mutant defective in copper transport, supporting the idea that the Ctr1 protein is itself a copper transporter; (2) transcription of ctr1 is detectable in P. ostreatus at all developmental stages and in all tissues (with the exception of lamellae), and is negatively regulated by the presence of copper in the culture medium; (3) ctr1 is a single-copy gene that maps to P. ostreatus linkage group III; and (4) the regulatory sequence elements found in the promoter of ctr1 are similar to those found in other copper-related genes described in other systems. These results provide the first description of a copper transporter in this white rot fungus and should be useful for further studies on copper metabolism in higher basidiomycetes.

A carbon–carbon-coupled dimeric bergenin derivative biotransformed by Pleurotus ostreatus

A novel C–C-coupled dimer derivative of bergenin was produced by the biotransformation of cultured mycelia of white rot fungus Pleurotus ostreatus. Its structure was elucidated by detailed spectroscopic analysis.

Structure selectivity in degradation and translocation of polychlorinated biphenyls (Delor 103) with a Pleurotus ostreatus (oyster mushroom) culture

The biodegradation of polychlorinated biphenyls (PCBs), formerly applied in technical mixtures called Delor 103, was realized by the white rot fungus Pleurotus ostreatus. Besides the efficiency of degradation the translocation of PCBs into the fruit bodies grown during cultivation on straw were of special interest. The selectivity of PCBs degradation is discussed on the basis of structural features of the individual congeners. The most important structural factors affecting the degradation efficiency are indicated with molecular descriptors. Basic dependences of the degradation efficiency of congeners on their chlorination grade and ortho chlorosubstitution could be proved. Additionally, other structural parameters such as the number and position of non-substituted carbon in the biphenyl molecule also affect the degradation results. Apart from the removal of Delor 103 the translocation of PCB isomers into the fruit bodies growing up during cultivation was observed. The structural features assisting the transport of PCB congeners from the straw substrate colonized with mushroom mycelium (straw/mycelium) to the mushroom fruit bodies are also relevant for the degradation mechanism but act reversely. Among the chlorination grade, structural parameters such as the number of para and adversely positioned chlorines or the number of occupied para and meta positions contribute to an accumulation of individual PCB congeners in the mushrooms. In total, only few PCB amounts (below 0.1% of the initially present PCBs in straw) were transported into the fruit bodies and a dangerous PCB contamination was never reached while the experiment. A combination of accelerated solvent extraction and GC-MS analysis was the analytical basis for the investigation presented.

Enzymatic decolorization of sulfonphthalein dyes

The white rot fungus (WRF) Pleurotus ostreatus produced manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities during solid state fermentation of wheat straw, a natural lignocellulosic substrate. Most of the sulfonphthalein (SP) dyes were decolorized by MnP at pH 4.0. The higher K m for meta-cresol purple (40 μM) and lower K m for ortho-cresol red (26 μM) for MnP activities explained the preference for the position of methyl group at ortho than at meta on chromophore. Bromophenol blue decolorizing activity was higher at pH 3.5 and decreased as the concentration of Mn II was increased. SP-decolorizing activity was associated not only with MnP but also with MIP. Additional bromine group along with the methyl group on SP chromophores decreases the rate of decolorization. Bromination of sulfonphthalein chromophore makes them the poorer substrate for MnP. This is evident from the higher K m for bromocresol green (117 μM) when compared to bromocresol purple (36 μM) and bromophenol blue (78 μM). The order of preference for the SP dyes as substrate for the MnP-catalyzed decolorizing activity is phenol red > ortho-cresol red > meta-cresol purple > bromophenol red > bromocresol purple > bromophenol blue > bromocresol green and the order of preference for the SP dyes as substrate for the MIP-catalyzed decolorizing activity is bromocresol green > bromophenol blue > bromocresol purple > bromophenol red > meta-cresol purple > ortho-cresol red > phenol red. Inhibition of PR decolorizing activity by NaN 3 provided the evidence of decolorizing activity as an oxidative process.

Production and induction of manganese peroxidase isozymes in a white-rot fungus Pleurotus ostreatus

The production of MnP by Pleurotus ostreatus in different liquid cultures was investigated. The highest level of activity was observed after 8 days of culture in peptone-glucose-yeast extract medium (PGY), whereas maximal activity was achieved after 30 days in glucose-yeast extract medium (GY). MnP was purified to homogeneity from PGY (designated MnP-PGY) and GY (MnP-GY). The isoelectric points of MnP-PGY and MnP-GY were 3.77 and 4.06, respectively. The molecular mass of both enzymes was 42 kDa. Analysis of the N-terminal amino acid sequence of purified MnPs and nucleotide sequence of cloned mnp indicated that MnP-GY has VTCATGQTTANE at the N-terminus, whereas MnP-PGY has ATCADGRTTANA. A putative exposed tryptophan residue (W170) was found in MnP-GY. Both isozymes oxidized veratryl alcohol, although the K(m) of MnP-GY was lower than that of MnP-PGY. Thus, the presence of peptone in the medium affected the production of MnP isozymes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the synthesis of MnP isozymes is controlled by culture conditions at the transcriptional level.

Reduced hardness as an indicator of susceptibility of timbers to attack by Euophryum confine Broun

Abstract The influence of early-wood hardness on the tunnelling activity of Euophryum confine Broun (Coleoptera: Curculionidae) is reported. The hardness of Pinus nigra (Arnold) sapwood blocks was measured using a micro-hardness testing device that simulated a mandible. Hardness was influenced by moisture content and wood decay. Hardness was found to be significantly lower at 25% equilibrium moisture content than at 15% in both undecayed wood and in wood decayed to 10% average weight loss by the brown rot fungi Coniophora puteana (Schum. Fr.) Karst., Serpula lacrymans (Wulf. Fr.) Schroet. and white rot fungus Pleurotus ostreatus (Jacq. Fr.) Kumm. In addition to this loss of weight from the blocks, there was also a significant reduction in early-wood hardness. Adult tunnelling was assessed over 7 days by wood weight loss, after any fungal decay had been arrested. Greater weight losses were recorded in blocks that had undergone fungal decay or had increased moisture content. Assessments of early-wood hardness in wild infestations of E. confine showed weevils tunnelled, decayed wood within an early-wood hardness range between 8.1 and 44.3 N / mm 2 .

The screening of culture condition and properties of xylanase by white-rot fungus Pleurotus ostreatus

Cultural conditions for xylanase production by Pleurotus ostreatus SYJ042 were screened in shake flask cultures. The results indicated that various factors including carbon and nitrogen source, air, pH, and inoculum size influence xylanase secretion. The optimum fermentation medium contained a carbon source (2.5% corn cob+2.5% wheat bran), nitrogen (0.8% peptone), inoculation level (four disks, 0.5 cm in diameter), aeration rate (1/3 fermentation liquid volume) and initial pH 6.0. Under these culture conditions, the maximum level of xylanase activity (24.98 U ml −1) was reached in 7 days. The enzyme was stable up to 40 °C, with 99.3% of the activity remaining after the enzyme had been incubated for 15 min. The enzyme appeared to be stable over a broad pH range (3.0–9.0) under assay conditions. The majority of the metal ions tested had little effect on enzyme activity with the exception of MnO 4 2− (strong inhibitor) and Ag + (modest inhibitor).

Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye

The white-rot fungus Pleurotus ostreatus strain 32 is an excellent producer of the industrially important enzyme laccase. Laccase was the only ligninolytic activity detected in the supernatant when the fungus was grown in liquid culture with or without shaking. Growth and laccase production in static cultivation were superior to that in agitated cultivation, and N-limited culture is of benefit to laccase production. When using cellobiose and peptone as carbon and nitrogen source, a higher activity level was obtained. 2,2′-Azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) (1 mM) was shown to be the best inducer of laccase production, reaching maximum values of about 400 U/ml. Cu 2+ (1 mM) also had a positive effect on laccase production, activity being enhanced to 360 U/ml. In addition, anthraquinone dye SN4R can be effectively decolorized by crude laccase (30 U/ml), the rate of which was 66%. The decolorization rate was increased by 90% with ABTS (0.16%) addition as a mediator of laccase.

Bioaugmentation of tar‐contaminated soils under field conditions using Pleurotus ostreatus refuse from commercial mushroom production

The influence of the white rot fungus Pleurotus ostreatus on the degradation of selected poly- and heterocyclic aromatic hydrocarbons (referred to as polycyclic aromatic hydrocarbons [PAHs]) in soil was investigated under field conditions representing the Northern temperate zone. Pleurotus ostreatus was added to two contaminated soils in the form of homogenized refuse from the commercial production of fungus. The soils were collected from a former shipyard (the B&W soil) and underneath a former coal tar storage at an old asphalt factory in Denmark (the Ringe soil). Treatments (control, soil mixed with autoclaved sawdust medium, and soil mixed with P. ostreatus refuse) were set up in triplicate in concrete cylinders (height, 50 cm; diameter, 60 cm). The activity of P. ostreatus was measured as laccase activity and phenanthrene (PHE)- and pyrene (PYR)-degrading bacteria were enumerated. Twenty-one different PAHs were quantified. After nine weeks the concentrations of the 3-, 4-, 5-, and 6-ring PAHs in the Ringe soil were reduced by 78, 41, and 4%, respectively. These reductions corresponded with high initial laccase activity, a decrease in pH caused by the fungus, and an increase in the number of PHE- and PYR-degrading bacteria. No significant PAH degradation was observed in the B&W soil. Reasons for the difference in performance of P. ostreatus in the two soils are discussed in terms of soil histories and bioavailability. The use of P. ostreatus refuse holds promising potential for bioremediation purposes.

Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium

Activities of cellulolytic and hemicellulolytic enzymes endo-1,4-β-glucanase, exo-1,4-β-glucanase, 1,4-β-glucosidase, endo-1,4-β-xylanase, 1,4-β-xylosidase and 1,4-β-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase were detected during the growth of the white-rot fungus Pleurotus ostreatus on wheat straw in the presence and absence of cadmium. The loss of substrate dry weight and Mn-peroxidase activity decreased with increasing Cd concentration, whereas the activities of endo-1,4-β-glucanase, 1,4-β-glucosidase and laccase were highly increased in the presence of metal. The onset of hemicellulose-degrading enzyme activity was delayed in the presence of cadmium. The degradation of a model synthetic dye Poly B-411 did not correspond to the activities of ligninolytic enzymes. This is the first report about 1,4-β-mannosidase in P. ostreatus.

Aryl alcohol oxidases from the white-rot basidiomycete Pleurotus ostreatus

Three aryl alcohol oxidases (AAOs; EC 1.1.3.7) I, II, and III from the culture filtrate of a strain of white-rot fungus Pleurotus ostreatus were purified by multistep chromatography. Each of the purified AAOs I, II, and III had the same molecular masses of 70kDa and 72kDa on gel filtration chromatography and sodium dodecyl sulfatepolyacrylamide gel electrophoresis, respectively. Their optimum temperature was 40°C, but their optimum pHs differed slightly. The N-terminal amino acid sequence of AAOs I, II, and III was determined to be Ala-Asp-Lys- Asp-Tyr-Ile-Val-Val-Gly-Ala, which showed significant similarity to those of Pleurotus eryngii (80% identity) and Pleurotus ostreatus Florida (60% identity)

Variability of laccase activity in the white-rot basidiomycetePleurotus ostreatus

The production of laccase in liquid cultures of the white-rot fungus Pleurotus ostreatus was highly variable. During the first days of cultivation, the relative variability was as high as 80-100% and it decreased to 30% in the course of cultivation. The main source of variability was assumed to be the independent development of enzyme activity in individual cultures. Cultures with high laccase production showed also high production of the other ligninolytic enzyme--Mn-dependent peroxidase. The variability was probably due to the source of inoculum, deactivation of the enzyme in culture liquid and genetic variations among the cultures. Variability of laccase activities was lower during solid-state fermentation on wheat straw and during the growth in nonsterile soil.

Removal of phenolics in olive mill wastewaters using the white-rot fungus Pleurotus ostreatus

Olive mill wastewaters (OMW) present a major environmental problem. The large amounts generated, combined with the high phenols and chemical oxygen demand concentrations, are the main difficulties in finding a solution for the management of these wastewaters, which are dangerous for the environment. The phenols, which are contained in the OMW have a structure similar to lignin, which makes them difficult to biodegrade. Lignin can be degraded only by a few microorganisms, such as “white-rot” basidiomycete, which produce manganese (MnPs) and lignin peroxidases (LiPs) and laccases that are responsible for the oxidisation of lignin compounds. The capability of Pleurotus ostreatus to degrade phenols of OMW in different conditions such as in sterilized and thermally processed (at 100°C) wastewater, with and without dilution, is investigated in this work. According to the experimental results P. ostreatus removed phenols from the culture medium, under all different conditions that were examined. The degradation of phenols reached up to 78.3% for the sterilized and 50% diluted OMW, 66.7% and 64.7% for the thermally processed OMW, with and without dilution, respectively. The effect of pre-treatment of OMW on the performance of anaerobic digestion is also assessed, as methanogenic bacteria are seriously affected by the presence of phenol compounds. The pre-treated wastewater was shown to be more amenable to a subsequent anaerobic digestion.

Degradation of ochratoxin a and b by the white rot funguspleurotus ostreatus

Degradation of ochratoxin A (OTA) and B (OTB) by three selected fungi during solid state fermentation of barley contaminated with ochratoxins was compared. In presence of the soil fungusRhizopus japonicus and the white rot fungusPanerochaete chrysosporium more than 60 % of the mycotoxins remained stable, while in the white rot fungusPleurotus ostreatus only 23 % of the initial OTA and 3 % of OTB were detected after a four weeks incubation period. Kinetic studies on mycotoxin degradation byPI ostreatus demonstrated formation of ochratoxin α and presumably ochratoxin β as intermediate products, what indicates that hydrolysis is the first step in OTA and OTB degradation followed by further degradation of the intermediates.

Mn2+ alters peroxidase profiles and lignin degradation by the white-rot fungus Pleurotus ostreatus under different nutritional and growth conditions.

The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn2+ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn2+ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of 14C-lignin mineralization was measured in the Mn2+-amended BSM, as determined during the course of 47 d of fermentation. Mn2+ amendment of the PM increased mineralization rate to that obtained in the Mn2+-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn2+ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn2+ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.

Copper and cadmium increase laccase activity in Pleurotus ostreatus.

Addition of copper (0.5-5 mM) or cadmium (1-5 mM) to the white rot fungus Pleurotus ostreatus cultivated in liquid nitrogen-limited medium for 12 days increased the activity of laccase. The addition of 2 mM Cd led to an 18.5-fold increase of activity, 1 mM Cu increased the activity eight-fold. When added earlier than 12 days, the activation of laccase was delayed (Cu) or decreased (Cd). Ag, Hg, Pb, Zn, and H(2)O(2) decreased laccase activity. To study the effect on native enzymes, purified laccase was incubated with Cd, Cu, and Hg. The addition of Hg decreased the activity of laccase immediately and reduced the temporal stability of the enzyme, while the addition of Cu (0.05-50 mM) increased both enzyme activity and stability. Laccase extracted at different stages of straw colonisation differed in its response to heavy metals.

Copper and cadmium increase laccase activity in Pleurotus ostreatus

Addition of copper (0.5–5 mM) or cadmium (1–5 mM) to the white rot fungus Pleurotus ostreatus cultivated in liquid nitrogen-limited medium for 12 days increased the activity of laccase. The addition of 2 mM Cd led to an 18.5-fold increase of activity, 1 mM Cu increased the activity eight-fold. When added earlier than 12 days, the activation of laccase was delayed (Cu) or decreased (Cd). Ag, Hg, Pb, Zn, and H 2O 2 decreased laccase activity. To study the effect on native enzymes, purified laccase was incubated with Cd, Cu, and Hg. The addition of Hg decreased the activity of laccase immediately and reduced the temporal stability of the enzyme, while the addition of Cu (0.05–50 mM) increased both enzyme activity and stability. Laccase extracted at different stages of straw colonisation differed in its response to heavy metals.

Transcript and activity levels of different Pleurotus ostreatus peroxidases are differentially affected by Mn2+.

The white-rot fungus Pleurotus ostreatus produces both manganese-dependent peroxidase (MnP) and versatile peroxidase (VP) in non-manganese-amended peptone medium (PM). We studied the effect of Mn2+ supplementation on MnPs and VPs in P. ostreatus by analysing the enzymatic and transcript abundance profiles of the peroxidases, as well as the lignin mineralization rate. The fungus was grown in PM under solid-state conditions using perlite as an inert solid support. Mn2+ amendment resulted in a 1.7-fold increase in [14C]-lignin mineralization relative to unamended medium. Anion-exchange chromatography was used to resolve the fungal peroxidase's enzymatic activity profile. Five peaks (P1-P5) of VP and one peak (P6) of MnP activity were detected in unamended medium. In Mn2+-amended medium, a reduction in the activity of the VPs was observed. On the other hand, a sharp increase in the MnP activity level of peak P6 was detected. The P6 isoenzyme was purified and showed manganese-dependent peroxidation of phenolic substrates. Internal sequence analysis of the purified enzyme revealed 100% identity with the deduced amino acid sequence of P. ostreatus MnP3 (GenBank AB016519). The effect of Mn2+ on the relative abundance of gene transcripts of three VPs and one MnP from P. ostreatus was monitored using reverse transcription-polymerase chain reaction (RT-PCR) with oligonucleotide primer sets synthesized on the basis of non-conserved sequences of the different peroxidases. The reduction in VP gene transcript abundance and the increase in mnp3 transcript level were collinear with the changes observed in the enzyme activity profiles. These results indicate that the activity of peroxidases is regulated at the transcriptional level. We suggest that the expression of MnP and VP may be differentially regulated by the presence of Mn2+.

PCB congener selective biodegradation by the white rot fungus Pleurotus ostreatus in contaminated soil

Six strains of white rot fungi were tested for their biodegradation ability of low chlorinated polychlorinated biphenyl (PCB) commercial mixture (Delor 103) in real soil system. Phanerochaete chrysosporium and Trametes versicolor did not show any ability to degrade PCBs in soil. On the contrary, four strains of Pleurotus ostreatus were able to remove about 40% of Delor 103 in two months. All P. ostreatus strains decomposed PCBs selectively with the preference for congeners with chlorine atoms in ortho > meta > para position. Degradation efficiency decreased with increasing number of chlorination.

Biodegradation of soil-adsorbed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus

The white rot fungus, Pleurotus ostreatus, metabolized four soil adsorbed polycyclic aromatic hydrocarbons: 50% of pyrene (0.1 mg g−1 dry soil), 68% of anthracene and 63% of phenanthrene were mineralized after 21 d. Biodegradation was increased to 75%, 80% and 75%, respectively of the initial concentration when 0.15% Tween 40 was added. Biodegradation of pyrene in the presence of surfactant and H2O2 (1.0 mM) was 90%. Benz[a]pyrene was also oxidized by Pleurotus ostreatus but it is not mineralized.