Isolation and purification of lignoperoxidase from the mushroom <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

The accumulation dynamics of lignolytic enzymes in culture media of the basidiomycetes Panus tigrinus, Pleurotus ostreatus, Fomes fomentarus, and the micromycete Aspergillus terreus were studied during the incubation period. It was found that Pleurotus ostreatus is the most active producer of lignoperoxidase enzymes among the studied fungi. Gel filtration and ion-exchange chromatography were used to isolate a homogeneous enzyme with lignoperoxidase activity. The maximum activity was found at pH 2.7 and 29°C. Gel electrophoresis determined the molecular weight (44 kDa). __________ Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 564–566, November–December, 2007.     

Solid-state fermentation of lignocellulosic plant residues from Brassica napus by Pleurotus ostreatus

Solid-state fermentation (SSF) of inedible parts of rapeseed was carried out using a white-rot fungus, Pleurotus ostreatus, to degrade lignocellulosic material for mycelial-single cell protein (SCP) production. This SSF system has the potential to be adapted to a controlled ecological life support system in space travel owing to the lack of storage space. The system for converting lignocellulosic material to SCP by P. ostreatus is simple; it can be carried out in a compact reactor. The fungal vegetative growth was better with a particle size of plant material ranging from 0.42 to 10 mm, whereas lignin degradation of the lignocellulose was the highest with particle sizes ranging from 0.42 to 0.84 mm. The addition of veratry alcohol (3,4-dimethoxybenzyl alcohol), hydrogen peroxide, and glycerol promotes lignocellulose degradation by P. ostreatus. The enhancement of bioconversion was also observed when a gas-flow bioreactor was used to supply oxygen and to maintain the constant moisture of the reactor. With this reactor, approx 85% of the material was converted to fungal and other types of biomass after 60 d of incubation.     

Co-cultured production of lignin-modifying enzymes with white-rot fungi

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

Biodegradation of Fluoranthene by Basidiomycetes Fungal Isolate <Emphasis Type="Italic">Pleurotus Ostreatus</Emphasis> HP-1

The biodegradation of fluoranthene, a high molecular weight polycyclic aromatic hydrocarbon (PAH), was investigated in submerged culture using the wood decaying fungus isolated from forest locality in Gujarat, India. The basidiomycete fungal isolate was found to have an ability to grow on sabaroud dextrose agar containing 50 mgl⁻¹ of each naphthalene, anthracene, acenaphthene, benzo (a) anthracene, pyrene, flouranthene, carbazole, and biphenyl. The involvement of extracellular fungal peroxidases such as manganese peroxidase (MnP) and laccase (Phenol oxidase) in the degradation of fluoranthene was studied. On the eighth day of incubation 54.09% of 70 mg l⁻¹ fluoranthene was removed. There after no PAHs removal was observed till the 20th day of the incubation period. The isolate was identified as Pleurotus ostreatus by 18S rRNA, 5.8S rRNA, and partial 28S rRNA gene sequencing. To the best of our knowledge this is the first time Pleurotus ostreatus have been reported to degrade such a high concentration of fluoranthene within much lower time period of incubation. Depletion in the residual fluoranthene in the culture medium was determined by HPLC. Attempts were made to identify the degradation product in the culture medium with the help of FT-IR, NMR, and HPTLC analysis. In the present study positive correlation between fluoranthene degradation and the ligninolytic enzyme (MnP and laccase) production is observed, thus this isolate can play an effective role for bioremediation of PAHs contaminated sites.     

Enzyme Production by Solid Substrate Fermentation of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Trametes versicolor</Emphasis> on Tomato Pomace

A process of solid state fermentation (SSF) on tomato pomace was developed with the white-rot fungi Pleurotus ostreatus and Trametes versicolor, using sorghum stalks as support. Operative parameters (humidity, water activity, and size of substrate particles) guaranteeing a good colonization of tomato pomace by both fungi were defined and conditions for production at high titers of the industrially relevant enzymes laccase, xylanase and protease were identified. Significant laccase activity levels (up to 36 U g⁻¹ dry matter) were achieved without any optimization of culture conditions, neither by nutrient addition nor by O₂ enrichment. Furthermore, protease activity levels up to 34,000 U g⁻¹ dry matter were achieved, being higher than those reported for the fungi typically considered as the best protease producers such as Aspergillus strains. Moreover, as one of the most significant results of this study, analysis of P. ostreatus tomato SSF samples by zymogram revealed two bands with laccase activity which had not been detected so far.     

TG–FTIR analysis applied to the study of thermal behaviour of some edible mushrooms

The article is devoted to the study on the thermal behaviour of three species of edible mushrooms: Boletus edulis (foot and cap), Pleurotus ostreatus (foot and cap), Lactarius deterrimus (cap) by the TG–FTIR-coupled technique, in air, over the 30–900 °C temperature range. The analysis of the TG–DTG–DTA curves reveals the thermal degradation mechanism to be complex and specific to every species under the recording conditions applied. A similar degradation mechanism is noticed for the foot and cap of Pleurotus ostreatus in comparison with the Boletus edulis and Lactarius deterrimus species where the mechanisms are different. The TG–FTIR analysis, combustion heats and IR spectra of the starting samples also support these results. The initial degradation temperatures from TG–DTG indicate the temperature range where these species are thermally stable and their nutrient features maintained making them proper for food. The TG–FTIR analysis gives information on the gaseous species evolved by the thermal degradation bringing thus a contribution to the elucidation of the changes developing by processing the edible mushrooms (industrialization, conservation, culinary preparations, etc.) at temperatures above the initial degradation temperature. At the same time, the environmental impact, when the mushroom failed cultures are burned, is also important.     

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.     

Solid state fermentation of plant residues for improved animal feed byPleurotus sp.

In laboratory-scale experiments, studies were made on the solid state fermentation of plant residues—rice straw and the upper soft portion of the stems of sarkanda (Saccharum munja)—by selected cultures of white-rot fungi,Pleurotus sajor-caju andPleurotus ostreatus. These cultures were selected after preliminary screening of their lignin-degrading capacities on lignin-agar medium. Their lignin degrading and (cellulose + hemicellulose) sparing, along with protein improving capacities, were studied for their potential application in animal feed production. A 100 g quantity of presoaked and sterilized residues was inoculated with wheat spawn of the two cultures and incubated at 25‡C. It was observed that, after 25 d, the crude protein contents (N × 6.25) of rice straw increased from 3 to 17.0% in the case of P.sajor-caju and to 19.2% in case of P. ostreatus. The percent removal values of cellulose, hemicellulose, and lignin were found to be as follows: 45.8, 16.8, and 47.1%, respectively, in the case ofP. sajor-caju and 56.5, 40.4, and 50%, respectively, in the case of P.ostreatus. After solid state fermentation of sarkanda for 25 d, its protein content increased from 3 to 12.8% in the case ofP. sajor-caju and to 14.5% in the case ofP. ostreatus. The percent removal of cellulose, hemicellulose, and lignin was found to be as follows: 31.2, 7.1, and 19%, respectively, in the case ofP. sajor-caju and 34.4, 7.1, and 14.3%, respectively, in the case ofP. ostreatus. The results obtained after solid state fermentation of the two residues by the mixed culture of these two basidiomycetes was also presented.     

Effect of copper and manganese ions on activities of laccase and peroxidases in three Pleurotus species grown on agricultural wastes

Copper (Cu²⁺) and manganese (Mn²⁺) ions influenced laccase (Lac) and peroxidase production in Pleurotus eryngii, Pleurotus ostreatus, and Pleurotus pulmonarius. In P. eryngii, the optimum Cu²⁺ concentration for Lac production was 1 mM and for peroxidases 10mM, and Mn²⁺ concentration of 5mM led to peaks of Lac and peroxidase activity. In P. ostreatus HAI 493, the highest level of Lac activity was at Cu²⁺ concentrations of 1 and 10 mM and Mn²⁺ concentration of 1mM, respectively. The absence of Cu²⁺ and Mn²⁺ caused the highest levels of peroxidase production. In P. ostreatus HAI 494, the highest level of Lac activity was at a Cu²⁺ concentration of 5 mM and at Mn²⁺ concentration of 1 mM, respectively. High levels of peroxidase activity were found in the medium without and with 1mM Cu²⁺, and at 1 and 5 mM Mn²⁺, respectively. In P. pulmonarius, the highest Lac activity was found in the presence of 5 mM Cu²⁺ and 5 mM Mn²⁺, respectively. The absence of Cu²⁺ and Mn²⁺ as well as their presence at a concentration of 1 mM led to the peaks of peroxidase activities.     

Comparison of volatile compound production in fruit body and in mycelium of Pleurotus ostreatus identified by submerged and solid-state cultures

Comparative analyses of the production of volatile compounds by Pleurotus ostreatus JMO.95 fruit body and its corresponding mycelium grown in liquid, on agar surface, and on solid support cultures were carried out by dynamic headspace concentration using gas chromatography/mass spectrometry and gas chromatography sniffing. The aroma produced by fruit body was owing essentially to the presence of octan-3-one and, to a lesser extent, to octan-3-ol. Other compounds, such as oct-1-en-3-ol, oct-1-en, 2-methylbutanol, and α-pinene were also present in low concentrations. Comparison of aromatic spectra of the fruit body with that of mycelia obtained under different culture conditions indicated that the main aromatic compounds present in the P. ostreatus fruit body and mycelium were produced in the same proportions on agar surface and on solid support culture, but not under submerged conditions.     

Enhanced Green Fluorescent Protein Expression in Pleurotus ostreatus for In Vivo Analysis of Fungal Laccase Promoters

The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus.     

Use of a Complete Factorial Experiment for Designing a Gas Sensor Based on Extracts of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> Mycelium Mushroom

A gas sensor was developed on the basis of extracts of mycelium mushroom Pleurotus ostreatus for determining phenol in air. The procedure of sensor preparation was optimized using a complete factorial experiment. The quantitative and kinetic parameters were found for the adsorption of some organic compounds (alcohols, ketones, alkyl acetates, phenols, aromatic amines) on films with a biological modifier.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 7, 2005, pp. 759–764.Original Russian Text Copyright © 2005 by Silina, Kuchmenko, Korenman, Tsivileva, Nikitina.     

Proteome Analysis and In Vitro Antiviral,Anticancer and Antioxidant Capacities of the Aqueous Extracts of Lentinula edodes and Pleurotus ostreatus Edible Mushrooms

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.     

Long-Term Monokaryotic Cultures of Pleurotus ostreatus var. florida Produce High and Stable Laccase Activity Capable to Degrade ß-Carotene

An extracellular laccase (Lacc10) was discovered in submerged cultures of Pleurotus ostreatus var. florida bleaching ß-carotene effectively without the addition of a mediator (650 mU/L, pH 4). Heterologous expression in P. pastoris confirmed the activity and structural analyses revealed a carotenoid-binding domain, which formed the substrate-binding pocket and is reported here for the first time. In order to increase activity, 106 basidiospore-derived monokaryons and crosses of compatible progenies were generated. These showed high intraspecific variability in growth rate and enzyme formation. Seventy-two homokaryons exhibited a higher activity-to-growth-rate-relation than the parental dikaryon, and one isolate produced a very high activity (1800 mU/L), while most of the dikaryotic hybrids showed lower activity. The analysis of the laccase gene of the monokaryons revealed two sequences differing in three amino acids, but the primary sequences gave no clue for the diversity of activity. The enzyme production in submerged cultures of monokaryons was stable over seven sub-cultivation cycles.

     

Facile Synthesis,Antimicrobial Activity and Molecular Docking of Novel 2,4,5‐Trisubstituted‐1H‐Imidazole–Triazole Hybrid Compounds

In the present work, a series of 18 imidazole–triazole hybrids ( 4a–r ) has been synthesized in good yield from substituted naphthaldehydes and 1,2‐diketones in the presence of ammonium acetate. The synthesized imidazole–triazole hybrid compounds were characterized by spectral techniques and screened in vitro for their antimicrobial activity. Compound 4h was found to be most active against Staphylococcus epidermidis, and compound 4e exhibited promising activity against Escherichia coli. In the fungal species under test, compound 4q was most potent against Aspergillus niger, even better than the fluconazole. Further, compound 4e was docked in the binding site of DNA gyrase topoisomerase II of E. coli.     

Edible Mushrooms as Source of Fibrin(ogen)olytic Enzymes: Comparison between Four Cultivated Species

Cardiovascular diseases represent the main cause of death. A common feature of cardiovascular disease is thrombosis resulting from intravascular accumulation of fibrin. In the last years, several fibrinolytic enzymes have been discovered in many medicinal or edible mushrooms as potential new antithrombotic agents. This study aimed to compare the fibrin(ogen)olytic activity of crude extracts from the fruiting bodies of four cultivated edible mushrooms: Lentinula edodes, Pleurotus ostreatus, Pleurotus eryngii, and Agrocybe aegerita. Fibrin(ogen)olytic activity was assessed by fibrin plate, spectrophotometric assay and electrophoretic analysis (SDS-PAGE and zymography). The highest activity was detected for P. ostreatus followed by P. eryngii, L. edodes and A. aegerita. Results indicated that enzymes exhibited maximum activity at pH 6–7 and 30–40 °C, respectively. Enzyme activity was inhibited by serine and metalloprotease inhibitors. We proposed a new index called the Specific Fibrin(ogen)olytic Index (SFI), which allows specification of the proportion of the total proteolytic capacity due to the fibrin(ogen)olytic activity. These data suggest that the extracts from fruiting bodies or powdered mushrooms can be used as functional ingredients for the development of new functional foods that may act as thrombolytic agents responding, at the same time, to the increasing demand for safe, healthy and sustainable food.     

Statistical Characterization of the Phytochemical Characteristics of Edible Mushroom Extracts

The antioxidant activity, total phenolic content, and total flavonoid content of 10 edible mushrooms species, including cultivated (Pleurotus ostreatus, Agaricus bisporus white and brown) and wild (P. ostreatus, Macrolepiota procera, Cantharellus cibarius, Russula vesca, Russula alutacea, Boletus edulis and Agaricus campestris), were determined. The extraction was performed using water and 50% water–ethanol and the caps and stipe were investigated separately. Water was the most appropriate solvent for phenolic compounds regardless of the sample. In contrast, the presence of ethanol in the solvent increased the extraction of flavonoids for cultivated P. ostreatus (caps and stipe), and wild R. alutacea, R. vesca, A. campestris, P. ostreatus (only caps) and C. cibarius, M. procera (only stipe). Significant differences between the antioxidant activities of the samples were registered in relationship with the different solvents. The antioxidant activity of water extract of dried A. bisporus brown (cultivated) showed the strongest 1,1-diphenyl-2-picrylhydrazyl radicals-scavenging assay (88.64%), while the B. edulis hydroalcoholic extract contained 74.93%. A detailed investigation into the functional group of phenolics and other organic compounds responsible with the antioxidant activity has been performed using Fourier transform infrared spectroscopy. The infrared spectra showed that the solvent-type directly influences the extraction process and, hence, the antioxidant activity. The present study contributes to information concerning mushrooms as sources of biologically active compounds. To investigate the correlations between phytochemical characteristics (i.e., phenolics, flavonoids and antioxidant activity) closely related to nature of solvents, the statistical analysis was performed.     

Influence of selected biowaste materials pre-treatment on their anaerobic digestion

The topic of this study is the pre-treatment of substrates for anaerobic digestion. Two different substrates of algae Scenedesmus subspicatus (SAG 86.81), Chlorella kessleri (LARG/1) and foliage of Prunus serrulata were subjected to anaerobic digestion. A mixture of commercially available cellulolytic enzymes (Analytical science s.r.o., Modra, Slovakia) was used for anaerobic treatment of algae while the foliage of Prunus serrulata was pre-treated by lignolytic fungi. The highest production of methane per mass of volatile solids was reached with untreated Chlorella kessleri at (0.59 ± 0.04) L g⁻¹. The addition of cellulolytic enzymes did not increase the production of methane from the algal substrate; however, a faster substrate degradation and thus also higher speed of methane production at the beginning of cultivation was achieved. After foliage pre-treatment by fungal isolate Pleurotus pulmonarius, isolated from natural habitats, the methane production increased five times. In this way we were able to speed up the processes of biological degradation of ligno-cellulose materials and thereby to increase the production of methane. Our results show the possibility of using algae as a suitable substrate for biogas production. On the other hand, also aerobic pre-treatment of foliage (Pleurotus pulmonarius) presents a successful way for speeding up the degradation of ligno-cellulose waste leading to increased methane yields.     

Tetraphenylporphyrin‐based dual‐functional medical agent for magnetic resonance and fluorescence imaging

A bimodal magnetic resonance imaging contrast agent, TPP‐M‐Gd, was developed by modifying tetraphenylporphyrin (TPP) with a small dendritic molecule as a ligand (M) to chelate gadolinium (Gd) ions. The ligand featured four carboxylate groups, which contributed to good water solubility and a strong combination with metal ions. The longitudinal relaxivity (R₁) of the resulting agent was calculated to be 12.45 mM^?1 s^?1, which is much higher than that of DTPA‐Gd (4.49 mM^?1 s^?1). The magnetic resonance imaging experiments showed that the newly synthesized contrast agent could enhance T₁‐weighted magnetic resonance imaging quality both in vitro and in vivo. In addition, TPP‐M‐Gd exhibited good fluorescent property as shown in cell imaging experiments. The cytotoxicity of TPP‐M‐Gd was even better than that of clinically approved DTPA‐Gd, which makes it a promising dual‐functional medical imaging agent to provide more detailed information about biological and disease‐related events.     

Bleach Enhancement of Mixed Wood Pulp by Xylanase–Laccase Concoction Derived Through Co-culture Strategy

Mixed enzyme preparation having both xylanase and laccase activity was evaluated for its bleach enhancing ability of mixed wood pulp. The enzyme was produced through co-cultivation of mutant Penicillium oxalicum SAU_E-3.510 and Pleurotus ostreatus MTCC 1804 under solid-state fermentation. Bleaching of pulp with mixed enzyme had resulted into a notable decrease in kappa number and increased brightness as compared to xylanase alone. Analysis of bleaching conditions had denoted that 8 IU g⁻¹ of mixed enzyme preparation (xylanase/laccase, 22:1) had led into maximal removal of lignin from pulp when bleaching was performed at 10% pulp consistency (55 °C, pH 9.0) for 3 h. An overall improvement of 21%, 8%, 3%, and 5% respectively in kappa number, brightness, yellowness, and viscosity of pulp was achieved under derived bleaching conditions. Process of enzymatic bleaching was further ascertained by analyzing the changes occurring in polysaccharide and lignin by HPLC and FTIR. The UV absorption spectrum of the compounds released during enzymatic treatment had denoted a characteristic peak at 280 nm, indicating the presence of lignin in released coloring matter. The changes in fiber morphology following enzymatic delignification were studied by scanning electron microscopy.