Gene cloning,heterologous expression,in vitro reconstitution and catalytic properties of a versatile peroxidase

The gene of a peroxidase described as being involved in carotenoid degradation was cloned from a strain that was conserved as Lepista irina (CBS 458.79). Gene sequencing revealed high nucleotide and amino-acid identity with Pleurotus eryngii gene vpl, which encodes a versatile peroxidase with unique catalytic properties, and only reported in Pleurotus and Bjerkandera species. Re-identification of the supposed L. irina strain revealed that, in fact, it is a P. eryngii strain. The new P. eryngii peroxidase was expressed in Escherichia coli, and the recombinant protein folded in the presence of cofactor to obtain the active form. The purified enzyme was able to oxidize Mn²⁺, veratryl alcohol, substituted phenols, and both low and high redox-potential dyes, demonstrating that it belongs to the versatile peroxidase family (named VPL3). These catalytic properties agreed with the presence of both Mn²⁺ and aromatic-substrate oxidation sites in its molecular structure.     

Identification of naphthalene metabolism by white rot fungus Pleurotus eryngii

The use of biomaterials or microorganisms in PAHs degradation had presented an eye-catching performance. Pleurotus eryngii is a white rot fungus, which is easily isolated from the decayed woods in the tropical rain forest, used to determine the capability to utilize naphthalene, a two-ring polycyclic aromatic hydrocarbon as source of carbon and energy. In the meantime, biotransformation of naphthalene to intermediates and other by-products during degradation was investigated in this study. Pleurotus eryngii had been incubated in liquid medium formulated with naphthalene for 14 days. The presence of metabolites of naphthalene suggests that Pleurotus eryngii begin the ring cleavage by dioxygenation on C1 and C4 position to give 1,4-naphthaquinone. 1,4-Naphthaquinone was further degraded to benzoic acid, where the proposed terepthalic acid is absent in the cultured extract. Further degradation of benzoic acid by Pleurotus eryngii shows the existence of catechol as a result of the combination of decarboxylation and hydroxylation process. Unfortunately, phthalic acid was not detected in this study. Several enzymes, including manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase and 2,3-dioxygenase are enzymes responsible for naphthalene degradation. Reduction of naphthalene and the presence of metabolites in liquid medium showed the ability of Pleurotus eryngii to utilize naphthalene as carbon source instead of a limited glucose amount.     

Polyethylene glycol-mediated transformation of fused egfp-hph gene under the control of gpd promoter in Pleurotus eryngii

Pleurotus eryngii was transformed using a polyethylene glycol-mediated method. A plasmid, pEPUGH, containing a reporter gene (enhanced green fluorescent protein gene, egfp) and a positive selectable marker gene (hygromycin phosphotransferase gene, hph) was constructed. The fused egfp-hph gene was placed under the control of the strong and constitutive native gpd promoter from P. eryngii. The recombinant plasmid was used to transform of P. eryngii protoplasts. Successful transformation was demonstrated by molecular analyses. Moreover, the mycelia of the transformants showed green epipolic dispersion on fluorescence microscopy. About 90–210 transformants were produced per μg plasmid DNA per 10⁷ viable protoplasts.     

PRODUCTION OF LIGNINOLYTIC ENZYMES BY SOLID-STATE FERMENTATION USING Pleurotus eryngii

Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ability to produce various ligninolytic enzymes such as laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) by solid-state fermentation (SSF), which was carried out using a support substrate from the fruit juice industry. The chemical content of grape waste from this industry was studied. Also, the production patterns of these extracellular enzymes were researched during the growth of the organism for a period of 20 days and the protein, reducing sugar, and nitrogen levels were monitored during the stationary cultivation. The highest Lac activity was obtained as 2247.62 ± 75 U/L on day 10 in the presence of 750 µM Mn²⁺, while the highest MnP activity was attained as 2198.44 ± 65 U/L on day 15 in the presence of 500 µM Mn²⁺. Decolorization of methyl orange and reactive red 2 azo dyes was also achieved with ligninolytic enzymes, produced in SSF of P. eryngii.     

Transformation of Industrial Dyes by Manganese Peroxidases from Bjerkandera adusta and Pleurotus eryngii in a Manganese-Independent Reaction

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn³⁺-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn²⁺. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn²⁺-independent manner. The reactions with the dyes were characterized by apparent K_m values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn²⁺, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn³⁺-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.     

Induction of Extracellular Hydroxyl Radical Production by White-Rot Fungi through Quinone Redox Cycling

A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe³⁺-EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺). H₂O₂ production, Fe³⁺ reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione [MD]). In all cases, OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H₂O₂ levels and Fe³⁺ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.White-rot fungi are unique in their ability to degrade a wide variety of organopollutants (36, 47), mainly due to the secretion of a low-specificity enzyme system whose natural function is the degradation of lignin (11). Components of this system include laccase and/or one or two types of peroxidase, such as lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP) (31). Besides acting directly, the ligninolytic enzymes can bring about lignin and pollutant degradation through the generation of low-molecular-weight extracellular oxidants, including (i) Mn³⁺, (ii) free radicals from some fungal metabolites and lignin depolymerization products (7, 22), and (iii) oxygen free radicals, mainly hydroxyl radicals (OH) and lipid peroxidation radicals (21). Although OH radicals are the strongest oxidants found in cultures of white-rot fungi (1), studies of their involvement in pollutant degradation are scarce. One of the reasons is that the mechanisms proposed for OH production still await in vivo validation.Several potential sources of extracellular OH based on the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺) have been postulated for white-rot fungi. In one case, an extracellular fungal glycopeptide has been shown to reduce O₂ and Fe³⁺ to H₂O₂ and Fe²⁺ (45). Enzymatic sources include cellobiose dehydrogenase, LiP, and laccase. Among these, only cellobiose dehydrogenase is able to directly catalyze the formation of Fenton''s reagent (33). The ligninolytic enzymes, however, act as an indirect source of OH through the generation of Fe³⁺ and O₂ reductants, such as formate (CO₂⁻) and semiquinone (Q⁻) radicals. The first time evidence was provided that a ligninolytic enzyme was involved in OH production, oxalate was used to generate CO₂⁻ in a LiP reaction mediated by veratryl alcohol (4). The proposed mechanism consisted of the following cascade of reactions: production of veratryl alcohol cation radical (Valc⁺) by LiP, oxidation of oxalate to CO₂⁻ by Valc⁺, reduction of O₂ to O₂⁻ by CO₂⁻, and a superoxide-driven Fenton reaction (Haber-Weiss reaction) in which Fe³⁺ was reduced by O₂⁻. The OH production mechanism assisted by Q⁻ was inferred from the oxidation of 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by Pleurotus eryngii laccase in the presence of Fe³⁺-EDTA. The ability of Q⁻ radicals to reduce both Fe³⁺ to Fe²⁺ and O₂ to O₂⁻, which dismutated to H₂O₂, was demonstrated (14). In this case, OH radicals were generated by a semiquinone-driven Fenton reaction, as Q⁻ radicals were the main agents accomplishing Fe³⁺ reduction. The first evidence of the likelihood of this OH production mechanism being operative in vivo had been obtained from incubations of P. eryngii with 2-methyl-1,4-naphthoquinone (menadione [MD]) and Fe³⁺-EDTA (15). Extracellular OH radicals were produced on a constant basis through quinone redox cycling, consisting of the reduction of MD by a cell-bound quinone reductase (QR) system, followed by the extracellular oxidation of the resulting hydroquinone (MDH₂) to its semiquinone radical (MD⁻). The production of extracellular O₂⁻ and H₂O₂ by P. eryngii via redox cycling involving laccase was subsequently confirmed using 1,4-benzoquinone (BQ), 2-methyl-1,4-benzoquinone, and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone), in addition to MD (16). However, the demonstration of OH production based on the redox cycling of quinones other than MD was still required.In the present paper, we describe the induction of extracellular OH production by P. eryngii upon its incubation with BQ, 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and MD in the presence of Fe³⁺-EDTA. The three benzoquinones were selected because they are oxidation products of p-hydroxyphenyl, guaiacyl, and syringyl units of lignin (MD was included as a positive control). Along with laccase, the involvement of P. eryngii VP in the production of O₂⁻ and H₂O₂ from hydroquinone oxidation has also been reported (13). Since hydroquinones are substrates of all known ligninolytic enzymes, quinone redox cycling catalysis could involve any of them. Here, we demonstrate OH production by P. eryngii under two different culture conditions, leading to the production of laccase or laccase and VP. We also show that quinone redox cycling is widespread among white-rot fungi by using a series of well-studied species that produce different combinations of ligninolytic enzymes.     

Versatile peroxidase as a valuable tool for generating new biomolecules by homogeneous and heterogeneous cross-linking

The modification and generation of new biomolecules intended to give higher molecular-mass species for biotechnological purposes, can be achieved by enzymatic cross-linking. The versatile peroxidase (VP) from Pleurotus eryngii is a high redox-potential enzyme with oxidative activity on a wide variety of substrates. In this study, VP was successfully used to catalyze the polymerization of low molecular mass compounds, such as lignans and peptides, as well as larger macromolecules, such as protein and complex polysaccharides. Different analytical, spectroscopic, and rheological techniques were used to determine structural changes and/or variations of the physicochemical properties of the reaction products. The lignans secoisolariciresinol and hydroxymatairesinol were condensed by VP forming up to 8 unit polymers in the presence of organic co-solvents and Mn²⁺. Moreover, 11 unit of the peptides YIGSR and VYV were homogeneously cross-linked. The heterogeneous cross-linking of one unit of the peptide YIGSR and several lignan units was also achieved. VP could also induce gelation of feruloylated arabinoxylan and the polymerization of β-casein. These results demonstrate the efficacy of VP to catalyze homo- and hetero-condensation reactions, and reveal its potential exploitation for polymerizing different types of compounds.     

Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species

Pleurotus eryngii, P. ostreatus and P. pulmonarius produced laccase (Lac) both under conditions of submerged fermentation (SF) and solid-state fermentation (SSF) with all of the investigated carbon and nitrogen sources. The highest levels of Lac activity were found in P. eryngii, under SF conditions of dry ground mandarine peels and in P. ostreatus, strain No. 493, under SSF conditions of grapevine sawdust.High levels of peroxidases activities were occurred in P. ostreatus, strain No. 494, and P. pulmonarius, under SSF conditions of grapevine sawdust, whereas in SF, these activities were either very low or absent.After purification of extracellular crude enzyme mixture of investigated species and strain which were grown in the medium with the best carbon sources, the Lac activity measurements revealed two peaks in P. eryngii, three peaks in both P. ostreatus strains and three in P. pulmonarius. Results obtained after purification also showed that the levels of phenol red oxidation in absence of external Mn²⁺ were higher than phenol red oxidation levels in presence of external Mn²⁺.In the medium with the best carbon sources (mandarine peels and grapevine sawdust, respectively), both P. eryngii and P. ostreatus, strain No. 493, showed the highest Lac activity with (NH₄)₂SO₄, as a nitrogen source, with a nitrogen concentration of 20 and 30 mM, respectively.In P. ostreatus, strain No. 494, and P. pulmonarius, the best nitrogen sources for peroxidases production were peptone in a concentration of 0.5% and NH₄NO₃ with a nitrogen concentration of 30 mM, respectively.     

Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans

Complementary DNA (cDNA) encoding the new versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii has been expressed in the ascomycete Emericella nidulans. In recombinant E. nidulans cultures, the pH reached values as high as 8.3, correlating with a sharp decrease in peroxidase activity. Peroxidase was rapidly inactivated at alkaline pH, but was comparatively stable at acidic pH. The peroxidase inactivation in alkaline buffer could be reversed by adding Ca²⁺ and lowering the pH. However, reactivation did not result after incubating the enzyme in non-buffered E. nidulans cultures that reached pH 7.5. To optimize recombinant peroxidase production, the effect of controlling the pH in E. nidulans bioreactor cultures was studied. An extended growth period, and a significant increase in the recombinant peroxidase level (5.3-fold higher activity than in the bioreactor without pH control) was obtained when the pH was maintained at 6.8, showing that culture pH is an important parameter for recombinant peroxidase production.     

Cultivation of different strains of king oyster mushroom (Pleurotus eryngii) on saw dust and rice straw in Bangladesh

Pleurotus eryngii is a popular mushroom due to its excellent consistency of cap and stem, culinary qualities and longer shelf life. In Bangladesh, where Pleurotus mushrooms are very popular, P. eryngii may take position among the consumers, but currently this mushroom is not cultivated in large scale there. In this study, 3 strains of P. eryngii such as Pe-1 (native to Bangladesh), Pe-2 (germplasm collected from China) and Pe-3 (germplasm collected from Japan) were cultivated on saw dust and rice straw and their growth and yield parameters were investigated. Pe-1 on saw dust showed the highest biological yield and efficiency (73.5%) than other strains. Also, the mycelium run rate and number of fruiting bodies were higher in Pe-1 than other two strains. The quality of mushroom strains was near about similar. On saw dust, the yield and efficiency were better than those cultivated on rice straw, however, on straw; the mushroom fruiting bodies were larger in size. This study shows the prospects of P. eryngii cultivation in Bangladesh and suggests further study in controlled environment for higher yield and production.     

Effect of culture temperature on the heterologous expression of <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> versatile peroxidase in <Emphasis Type="Italic">Aspergillus</Emphasis> hosts

Production of recombinant versatile peroxidase in Aspergillus hosts was optimized through the modification of temperature during bioreactor cultivations. To further this purpose, the cDNA encoding a versatile peroxidase of Pleurotus eryngii was expressed under control of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans. A dependence of recombinant peroxidase production on cultivation temperature was found. Lowering the culture temperature from 28 to 19 °C enhanced the level of active peroxidase 5.8-fold and reduced the effective proteolytic activity twofold. Thus, a maximum peroxidase activity of 466 U L^-1 was reached. The same optimization scheme was applied to a recombinant Aspergillus niger that bore the alcohol dehydrogenase regulator (alcR), enabling transformation with the peroxidase cDNA under the same alcA promoter. However, with this strain, the peroxidase activity was not improved, while the effective proteolytic activity was increased between 3- and 11-fold compared to that obtained with A. nidulans.     

Functional expression of bovine growth hormone gene in Pleurotus eryngii

The expression of the bovine growth hormone (bGH) gene was examined in Pleurotus eryngii, which belongs to the family of oyster mushrooms. The region encoding mature bGH, which has a variety of regulatory effects on growth and metabolic processes, was amplified using designed primers containing initiation and termination codons and then subcloned into pPEV binary expression vector. The recombinant vector (pPEVbGH) was introduced in P. eryngii via Agrobacterium tumefaciens-mediated transformation. Recombinant bGH was expressed in P. eryngii harboring pPEVbGH vector under control of the cauliflower mosaic virus (CaMV) 35S promoter up to a level of approximately 26% of total cell proteins after 6 days of cultivation, after which the recombinant protein was analyzed by SDS-PAGE and Western blotting. Interestingly, the growth rate of P. eryngii mycelia harboring pPEVbGH vector was approximately three times faster than that of control P. eryngii, suggesting that bGH affected the growth of P. eryngii. Biological activities were examined in Sprague-Dawley rats, which were administered regular feed mixed with mycelial extracts containing bGH (0.1 or 0.2 μg of bGH per g of animal feed). Mycelial extracts containing bGH significantly affected growth rates and lipid profiles; total cholesterol, triglyceride, HDL, and LDL levels were improved in rats fed mycelial extracts compared with those administered regular feed containing nontransgenic P. eryngii. This result indicates that P. eryngii harboring pPEVbGH vector could produce biologically active bGH. Further, levels of all growth-related factors increased, resulting in faster growth rates in bGH-treated groups. Accordingly, these data suggest that P. eryngii can be applied to the production of industrially useful proteins using a plant expression vector as an efficient mushroom host system.     

Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS,DIRECTED MUTAGENESIS,EPR, AND NMR STUDY*

Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn²⁺, and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H₂O₂-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan.     

Genetic Variability and Population Structure of the Mushroom Pleurotus eryngii var. tuoliensis

The genetic diversity of 123 wild strains of Pleurotus eryngii var. tuoliensis, which were collected from nine geographical locations in Yumin, Tuoli, and Qinghe counties in the Xinjiang Autonomous Region of China, was analysed using two molecular marker systems (inter-simple sequence repeat and start codon targeted). At the variety level, the percentage of polymorphic loci and Nei’s gene diversity index for P. eryngii var. tuoliensis was 96.32% and 0.238, respectively. At the population level, Nei’s gene diversity index ranged from 0.149 to 0.218 with an average of 0.186, and Shannon''s information index ranged from 0.213 to 0.339 with an average of 0.284. These results revealed the abundant genetic variability in the wild resources of P. eryngii var. tuoliensis. Nei’s gene diversity analysis indicated that the genetic variance was mainly found within individual geographical populations, and the analysis of molecular variance revealed low but significant genetic differentiation among local and regional populations. The limited gene flow (Nm = 1.794) was inferred as a major reason for the extent of genetic differentiation of P. eryngii var. tuoliensis. The results of Mantel tests showed that the genetic distance among geographical populations of P. eryngii var. tuoliensis was positively correlated with the geographical distance and the longitudinal distances (r_Go = 0.789 and r_Ln = 0.873, respectively), which indicates that geographical isolation is an important factor for the observed genetic differentiation. Nine geographical populations of P. eryngii var. tuoliensis were divided into three groups according to their geographical origins, which revealed that the genetic diversity was closely related to the geographical distribution of this wild fungus.     

Evaluation of Burma Reed as Substrate for Production of Pleurotus eryngii

Burma reed (Neyraudia reynaudiana), a giant C₄ grass, was included in substrate at the rates of 0, 20, 40 and 66 % to partially or wholly substitute sawdust and cottonseed hulls to evaluate its suitability for Pleurotus eryngii cultivation. Inclusion of 20 and 40 % Burma reed did not significantly affect linear mycelial growth, dry matter loss, spawn run period and fructification, and achieved high fruiting body yields and biological efficiency of 336.67 g/bag, 67.33 % and 342.15 g/bag, 68.43 %, respectively, which were not significantly different from 350.08 g/bag to 70.02 % obtained from the control substrate. Enzyme assay revealed that on the mixed substrates laccase and manganese peroxidase activity were significantly enhanced, but cellulase was significantly reduced in the middle stage of incubation as compared with the control substrate. Even on Burma reed substrate without sawdust and cottonseed hulls, fruiting body yield (313.56 g/bag) and biological efficiency (62.71 %) were satisfactory, although significantly lower than that on the control substrate. Therefore, Burma reed was a promising potential substrate for P. eryngii production to largely substitute sawdust and cottonseed hulls.     

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H₂O₂-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw.     

Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization

The main manganese peroxidase (MnP) isoenzyme of Agaricus bisporus ATCC 62459 produced in lignocellulose-containing cultures was isolated, cloned and sequenced. In liquid medium, where MnP was previously detected only in trace amounts, the production of MnP was enhanced by rye and wheat bran supplements. The pI (3.25) and N-terminal amino acid sequence (25 aa) of the enzyme from bran-containing cultures were identical to those reported from compost-isolated MnP1. MnP1 is a 328-aa long polypeptide preceded by a 26-aa leader peptide. The nucleotide sequence and putative amino acid sequence of MnP1 reveal its similarity to Pleurotus ostreatus MnP3 (62.5%), Lepista irina versatile peroxidase (VP) (61.8%) and Pleurotus eryngii VPs VPL2 and VPL1 (61.9% and 61.2%, respectively). The intron-exon structure resembles that of P. ostreatus MnP1 and P. eryngii VPL1. Despite the sequence similarity to VPs, in the A. bisporus MnP1 sequence, alanine (A163) is present instead of tryptophane (W164), distinguishing it from the veratryl alcohol oxidising P. eryngii VPLs. The MnP sequence can be used as a tool to examine the pattern of ligninolytic gene expression during the growth and fruiting of A. bisporus to optimise compost composition, fungal growth and mushroom production.     

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> complex

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man’s selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods—amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes—to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.     

Prediction of hydrogen-bonding networks around tyrosyl radical in P. eryngii versatile peroxidase W164Y variants: a QM/MM MD study

Quantum mechanics/molecular mechanics molecular dynamics simulations have been used to predict the hydrogen-bonding networks in the active site of three double variants of Pleurotus eryngii versatile peroxidase containing a redox-active tyrosine in place of a tryptophan residue in position 164. The adopted computational strategy has proved to be adequate to correctly reproduce the hydrogen-bonding environment of tyrosyl radical (Tyr^√) in the single W164Y variant of the enzyme that has been directly identified by electron paramagnetic resonance spectroscopy. In this study, we have investigated the effect of the mutation of a specific amino acid in the local environment of tyrosine 164. We show that the substitution of arginine 257 with a glutamic acid, a leucine or an alanine residue is able to induce the stabilisation of different hydrogen-bonding networks involving Tyr^√ that can potentially affect its physico-chemical properties.