Edible Mushroom Pleurotus Ostreatus Research Articles

Toxic and lethal effects of ostreolysin, a cytolytic protein from edible oyster mushroom ( Pleurotus ostreatus), in rodents

Ostreolysin (Oly), an acidic, 15 kDa protein from the edible oyster mushroom ( Pleurotus ostreatus), is a toxic, pore-forming cytolysin. In this paper, its toxic properties have been studied in rodents and the LD 50 in mice shown to be 1170 μg/kg. Electrocardiogram, arterial blood pressure and respiratory activity were recorded under general anaesthesia, in intact, pharmacologically vagotomised and artificially respirated rats injected with one mouse LD 50. A few seconds after intravenous Oly injection, a transient increase in arterial blood pressure was recorded, followed by a progressive fall to mid-circulatory pressure accompanied by bradicardia, myocardial ischaemia and ventricular extrasystoles. Similar changes produced by Oly were observed in vagotomised and artificially respirated animals, indicating that vagotomy and hypoxia play no primary role in toxicity. Oly induced lysis of rat erythrocytes in vitro, and probably also in vivo as indicated by the increase in serum potassium. Although direct action of the protein on the cardiomyocytes or heart circulation cannot be excluded, the hyperkalaemia resulting from the haemolytic activity probably plays an important role in its toxicity. The lethality and cardiorespiratory toxic action of Oly are thus shown to be candidates for the cause of the recorded adverse effects of oyster mushroom.

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

Ostreolysin, a cytolytic protein from the edible oyster mushroom ( Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.

An aqueous polysaccharide extract from the edible mushroom Pleurotus ostreatus induces anti-proliferative and pro-apoptotic effects on HT-29 colon cancer cells

Anti-proliferative and pro-apoptotic activities of fractions of Pleurotus ostreatus were examined using HT-29 colon cancer cells in vitro. A hot-water-soluble fraction of the mycelium of the liquid cultured mushroom was partially isolated and chemically characterized as a low-molecular-weight alpha-glucan. HT-29 cells were exposed to the different isolates and significant inhibition of proliferation was obtained in a dose-dependent manner. Proliferation inhibition was shown to be the result of apoptotic induction because the pro-apoptotic molecules Bax and cytosolic cytochrome-c were upregulated. Fluorescence-activated cell sorter analyses of polysaccharide-treated HT-29 cells showed a high percentage of Annexin-positive cells. Here, we describe a newly identified low-molecular-weight alpha-glucan with promising anti-tumorigenic properties, and demonstrate its direct effect on colon cancer cell proliferation via induction of programmed cell death.

Strain discrimination of an edible mushroom Pleurotus ostreatus by DNA technology

Strain discrimination of an edible mushroom Pleurotus ostreatus by DNA technology

Response Surface Analysis of Solid State Growth of Pleurotus ostreatus Mycelia utilizing Whey Permeate

A novel approach to utilizing whey permeate, the cultivation of mycelia of the edible mushroom Pleurotus ostreatus, is introduced. Response surface analysis (RSA) was successfully applied to determine the combination of substrate concentration, temperature and pH that would result in a maximal mycelial extension rate under solid state cultivation. The conditions to maximize the mycelial extension rate were predicted to be 44 g lactose l(-1), pH 6.0 and 24.2 degrees C. Subsequent verification of these levels agreed with model predictions.

Effect of pH on the Pore Forming Activity and Conformational Stability of Ostreolysin, a Lipid Raft-Binding Protein from the Edible MushroomPleurotus ostreatus,

Ostreolysin, a pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is a member of the aegerolysin protein family, a novel group of small acidic proteins found in bacteria, molds, mushrooms, and plants. It binds to lipid rafts and interacts specifically with cholesterol-rich lipid domains. In this study, ostreolysin was classified as a single-domain all-beta-structured protein on the basis of cDNA sequencing. pH-induced and thermally induced unfolding of ostreolysin was studied by means of CD, UV absorption, and intrinsic tryptophan fluorescence to characterize conformational transitions associated with its functional properties, i.e., binding to lipid membranes, pore forming activity on lipid vesicles, and hemolysis. At 25 degrees C and between pH 6 and 9, ostreolysin adopted a monomeric and thermodynamically stable nativelike conformation, characterized by rigid tertiary structure and predominantly beta-sheet secondary structure. Between pH 2 and 3, the protein underwent an irreversible transition to a partially unfolded, molten globule-like state which bound ANS, and exhibited disrupted tertiary structure and enhanced non-native alpha-helical structure. Functional studies showed that, unlike colicins and some other bacterial pore-forming toxins, the acid-induced molten globule-like state of ostreolysin is not relevant for lipid binding and pore formation. Instead, the compact native state was necessary for binding to cholesterol/sphingomyelin multilamellar vesicles, optimally in the pH range from 6 to 7, and for pore formation and hemolysis, maximally between pH 7 and 8.

The use of spectral mapping for the study of the enzyme production of the edible mushroom Pleurotus Ostreatus

The effect of the addition of extracts of agro-industrial wastes to the culture media on the production of s-glucosidase, xylanase, laccase, manganese-dependent and independent peroxidases by the edible fungus Pleurotus ostreatus was determined. The relationship between cultivation parameters and the enzyme activities was assessed by spectral mapping technique combined with non-linear mapping. It was proved that extracts enhanced markedly the activities of laccase, manganese-dependent and independent peroxidases. Multivariate mathematical-statistical methods indicated that the enzyme activities were the highest in culture containing pepper extract. It was further demonstrated that the selectivity of the enzyme production was negligible up till 14 days of fermentation and reached the maximum at the 28 th day.

Enzyme Production of the Edible Mushroom Pleorotus ostreatus in Shaken Cultures Completed with Agro‐Industrial Wastes

AbstractThe enzyme production of the white‐rot fungus, the edible mushroom Pleurotus ostreatus, was determined in shaken culture media containing extracts of agro‐industrial wastes (tomato, potato and pepper residues) as an unique carbon source. The activity of β‐glucosidase, xylanase, laccase as well as manganese‐dependent and independent peroxidases was measured at 0, 3.5, 7.0, 10.5, 14.0, 17.5, 21.0, 24.5, 28.0 and 31.5 days of cultivation. A spectral mapping technique and non‐linear mapping were employed for the calculation of the relationships among the fermentation parameters, such as fermentation time, enzyme activity and selectivity of enzyme production. It was established that P. ostreatus produced β‐glucosidase, xylanase, laccase, manganese‐dependent and independent peroxidases in each culture medium and that the enzyme activities were higher in cultures containing agro‐industrial wastes than in the control containing glucose as a carbon source. The spectral mapping technique allowed demonstrating that the enzyme activities were the highest in the culture completed with pepper extract followed by cultures containing potato and tomato extracts. The differences among the selectivity of the enzyme activities were negligible up to 21.0 days of fermentation and reached the maximum at the end of the fermentation process. The production of laccase as well as manganese‐dependent and independent peroxidases showed similar patterns while the selectivity patterns of xylanase and β‐galactoside production were different. In addition, it became evident that the agro‐industrial wastes influenced the enzyme production in a distinct way.

Ostreolysin, a pore-forming protein from the oyster mushroom, interacts specifically with membrane cholesterol-rich lipid domains

Ostreolysin, a 15 kDa pore-forming protein from the edible oyster mushroom ( Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their cholesterol contents and with the occurrence of ostreolysin in the cells detergent resistant membranes. Moreover, ostreolysin binds to supported monolayers and efficiently permeabilizes sonicated lipid vesicles, only if cholesterol is combined with either sphingomyelin or dipalmitoylphosphatidylcholine. Addition of mono- or di-unsaturated phosphatidylcholine to the cholesterol/sphingomyelin vesicles dramatically reduces the ostreolysin's activity. It appears that the protein recognizes specifically a cholesterol-rich lipid phase, probably the liquid-ordered phase.

Pleurotolysin, a Novel Sphingomyelin-specific Two-component Cytolysin from the Edible Mushroom Pleurotus ostreatus, Assembles into a Transmembrane Pore Complex

Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8-5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25 degrees C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.

Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus

Pleurotolysin, a sphingomyelin-specific cytolysin consisting of A (17 kDa) and B (59 kDa) components from the basidiomycete Pleurotus ostreatus, assembles into a transmembrane pore complex. Here, we cloned complementary and genomic DNAs encoding pleurotolysin, and studied pore-forming properties of recombinant proteins. The genomic regions encoding pleurotolysin A and B contained two and eight introns, respectively, and putative promoter sequences. The complementary DNA (cDNA) for pleurotolysin A encoded 138 amino acid residues, and the predicted product was identical with natural pleurotolysin A, except for the presence of the first methionine. Recombinant pleurotolysin A lacking the first methionine was purified as a 17-kDa protein with sphingomyelin-binding activity. The cDNA for pleurotolysin B encoded a precursor consisting of 523 amino acid residues, of which N-terminal 48 amino acid residues were absent in natural pleurotolysin B. Mature and precursor forms of pleurotolysin B were expressed as insoluble 59- and 63-kDa proteins, respectively, which were unfolded with 8 M urea and refolded by 100-fold dilution with 10 mM Tris–HCl buffer, pH 8.5. Although neither recombinant pleurotolysin A nor B alone was hemolytically active at higher concentrations of up to 100 mg/ml, they cooperatively assembled into a membrane pore complex on human erythrocytes and lysed the cell as efficiently as the natural proteins at nanomolar concentrations. In contrast, the precursor of pleurotolysin B was much less hemolytically active than mature pleurotolysin B in the presence of pleurotolysin A.

Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipids.

Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes, we compared its effects on mammalian cells, on vesicles prepared with either pure lipids or total lipid extracts, and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations, the protein lysed human, bovine and sheep erythrocytes by a colloid-osmotic mechanism, compatible with the formation of pores of 4 nm diameter, and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids, occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact, permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes, and not with lipids extracted from P. ostreatus or pure lipid mixtures, even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization, correlated with an increase in the intrinsic fluorescence and alpha-helical content of the protein, and with aggregation, which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting.

Purification and characterization of a new ribonuclease from fruiting bodies of the oyster mushroom Pleurotus ostreatus

A ribonuclease (RNase), possessing an N-terminal sequence disparate from those of ribonucleases from other mushrooms and previously isolated Pleuotus ostreatus RNases, was purified from the fruiting bodies of the edible mushroom Pleurotus ostreatus. The N-terminal sequence of Pleurotus ostreatus RNase did not manifest homology even to a previously reported RNase from the same mushroom. The ribonuclease was adsorbed on CM-Sepharose and Mono S. It exhibited a molecular mass of 12 kDa in both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. The ribonuclease displayed an activity of 11490 U/mg on yeast tRNA. The highest ribonuclease activity was exhibited toward poly U, followed by poly A and poly C. No activity was shown toward poly G. The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C. It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 240 nM.

Lignocellulolytic Enzyme Activity During Growth and Fruiting of the Edible and Medicinal Mushroom Pleurotus ostreatus (Jacq.:Fr.) Kumm. (Agaricomycetideae)

Cotton wastes can be converted without substrate pasteurization into value-added products, such as gourmet mushrooms, with biological efficacy up to 90% and lignocellulolytic enzymes. Edible and medicinal mushroom Pleurotus ostreatus carboxymethyl cellulase and laccase activities, extracted from both the surface and inside layers of blocks, increased during primordia and fruiting bodies development and declined rapidly after the harvest. In a study of xylanase production, it has been observed that the activity of the enzyme in inside layers of the substrate gradually increased during the 49 days (second fruiting stage) after inoculation and then gradually declined towards the end of mushroom cultivation. Fluctuations in xylanase activity in the surface layer were observed, so that maximal enzyme activities coincided with the fruiting stage of P. ostreatus development. In contrast to laccase, manganese peroxidase (MnP) activity was high during the colonization stage and declined during the first primordia and fruiting body formation stages. After this, while laccase activity decreased, MnP activity peaked at the mycelia stage in oyster mushroom development. Data on the levels of cellulase, xylanase, laccase, and MnP activity in spent mushroom substrate demonstrate that it can be used as an excellent source of these enzymes.

Purification and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus

Nineteen fungi were tested for their ability to degrade aflatoxin B1 (AFB1). An extracellular enzyme from the edible mushroom Pleurotus ostreatus showed afaltoxin-degradation activity detected by thin-layer chromatography (TLC). An enzyme with this activity was purified by two chromatographies on DEAE-Sepharose and Phenyl-Sepharose. The apparent molecular mass of the purified enzyme was estimated to be 90 kDa by SDS-PAGE. Optimum activities were found in the pH range between 4.0 and 5.0 and at 25 degrees C. Also, degradation activity of several dyes in the presence of H2O2 was tested, resulting in the detection of bromophenol blue-decolorizing activity. Based on these data, we suggest this enzyme is a novel enzyme with aflatoxin-degradation activity. Fluorescence measurements suggest that the enzyme cleaves the lactone ring of aflatoxin.

Transformation of the edible mushroom Pleurotus ostreatus by particle bombardment

Uracil auxotroph of Pleurotus ostreatus was transformed to prototrophy by means of particle bombardment. Five transformants were obtained under three conditions differing in the two parameters of target distance and helium pressure. The transformation frequency was one transformant per μg of DNA. In the transformants, plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of P. ostreatus by particle bombardment.

Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg 2+ but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.

A Lectin from the Edible and Medicinal Mushroom Pleurotus ostreatus (Jacq.: Fr.) Kumm. as a Food Intake Suppressing Substance

A Lectin from the Edible and Medicinal Mushroom Pleurotus ostreatus (Jacq.: Fr.) Kumm. as a Food Intake Suppressing Substance

A New Lectin with Highly Potent Antihepatoma and Antisarcoma Activities from the Oyster Mushroom Pleurotus Ostreatus

A dimeric lectin, composed of subunits with a molecular weight of 40 and 41 kDa, respectively, and demonstrating similarity in N-terminal sequence to each other and to Aleuria aurantia lectin, was isolated from fresh fruiting bodies of the edible mushroom Pleurotus ostreatus. The lectin was unadsorbed on DEAE-cellulose in 10 mmol/L phosphate buffer (pH 7.5), subsequently adsorbed on CM-cellulose in 10 mmol/L NH4OAc (pH 5.4), and came off in the first peak from a Superose 12 column during fast protein liquid chromatography. The lectin was acid-labile, alkali-labile, and heat-labile. Its hemagglutinating activity was sensitive to inhibition by CaCl2, MgCl2, MnCL2 and FeCl3 and potentiation by AlCl3. Melibiose, lactose, d-galactose, α-methyl-d-galactopyranoside, N-acetylneuraminic acid, raffinose, and inulin were capable of inhibiting its hemagglutinating activity, with melibiose being the most potent. The lectin exerted potent antitumor activity in mice bearing sarcoma S-180 and hepatoma H-22. Survival in these mice was prolonged and body weight increase reduced after lectin treatment.

A lectin from an edible mushroom Pleurotus ostreatus as a food intake-suppressing substance

In an experiment in which rats were allowed free access to food and water, the rats did not eat the diet containing a mushroom Pleurotus ostreatus even if they were emaciated. A P. ostreatus lectin (POL) was isolated from the mushroom as the food intake-suppression principle. In hemagglutination inhibition assays, Me-αGalNAc was the most potent inhibitor among the monosaccharides tested. Among all the sugars tested, 2′-fucosyllactose (Fucα1→2Galβ1→4Glc) was the strongest inhibitor and its inhibitory potency was five times greater than that of Me-αGalNAc. POL exhibited a binding ability to bovine submaxillary mucin (BSM) and asialo-BSM and the other glycoproteins were inert to the binding. The food intake-suppressing activity of POL was dependent on the dose. The diet containing 0.1% POL caused a 50% decrease in the food intake of rats against the control.