Mycoremediation of oil contaminant by Pleurotus florida (P.Kumm) in liquid culture.

This study investigated the potential functions of Pleurotus florida (an edible mushroom) in the biodegradation of gas oil at concentrations of 0 (control), 2.5, 5, and 10% (V: V) for 30 days. The gas oil increased dry weight and protein concentration in all treatments (by an average of 19.5 and 108%, respectively). Moreover, the pH, surface tension (ST), and interfacial tension (IFT) were reduced by the mushroom supplementation. The lowest surface tension (31.9 mN m-1) and the highest biosurfactant production belonged to the 10% gas oil treatment (0.845 ± 0.03 mg mL-1). The results demonstrated that the adsorption isotherm agreed well with the Langmuir isotherm. The maximum Langmuir adsorption capacity was calculated at 0.743 mg g-1 wet biomass of P. florida. The fungal supplementation efficiently remedied the total petroleum hydrocarbons (TPHs) by an average of 55% after 30 days. Gas chromatography (GC) analysis revealed that P. florida effectively detoxified C13-C28 hydrocarbons, Pristane, and Phytane, implying its high mycoremediation function. The toxicity test showed that mycoremediation increased the germination by an average of 35.82% ± 8.89 after 30 days. Laccase activity increased significantly with increasing gas oil concentration in the treatments. The maximum laccase activity was obtained in the 10% gas oil treatment (142.25 ± 0.72 U L-1). The presence of pollutants was also associated with induction in the tyrosinase activity when compared to the control. These results underline the high mycoremediation capacity of P. florida through the involvement of biosurfactants, laccase, and tyrosinase.

Upcycling the Spent Mushroom Substrate of the Grey Oyster Mushroom Pleurotus pulmonarius as a Source of Lignocellulolytic Enzymes for Palm Oil Mill Effluent Hydrolysis.

Mushroom cultivation along with the palm oil industry in Malaysia have contributed to large volumes of accumulated lignocellulosic residues that cause serious environmental pollution when these agroresidues are burned. In this study, we illustrated the utilization of lignocellulolytic enzymes from the spent mushroom substrate of Pleurotus pulmonarius for the hydrolysis of palm oil mill effluent (POME). The hydrolysate was used for the production of biohydrogen gas and enzyme assays were carried out to determine the productivities/activities of lignin peroxidase, laccase, xylanase, endoglucanase and ß-glucosidase in spent mushroom substrate. Further, the enzyme cocktails were concentrated for the hydrolysis of POME. Central composite design of response surface methodology was performed to examine the effects of enzyme loading, incubation time and pH on the reducing sugar yield. Productivities of the enzymes for xylanase, laccase, endoglucanase, lignin peroxidase and ß-glucosidase were 2.3, 4.1, 14.6, 214.1, and 915.4 U g-1, respectively. A maximum of 3.75 g/l of reducing sugar was obtained under optimized conditions of 15 h incubation time with 10% enzyme loading (v/v) at a pH of 4.8, which was consistent with the predicted reducing sugar concentration (3.76 g/l). The biohydrogen cumulative volume (302.78 ml H2.L-1 POME) and 83.52% biohydrogen gas were recorded using batch fermentation which indicated that the enzymes of spent mushroom substrate can be utilized for hydrolysis of POME.

A water-soluble selenium-enriched polysaccharide produced by Pleurotus ostreatus: Purification, characterization, antioxidant and antitumor activities in vitro.

The development and application of new selenium-enriched polysaccharides has become a critical topic in recent years. In this study, a natural selenium-enriched polysaccharide fraction (Se-POP-21) produced by Pleurotus ostreatus was purified, characterized, and investigated the antioxidant and antitumor activities in vitro. The Se-POP-21 was mainly composed of mannose, glucose, galactose and arabinose, with a molar ratio of 18.01:2.40:26.15:7.34, of which molecular weight was 15,888 Da and the selenium content was 5.31 µg/g. Spectral analysis demonstrated that Se-POP-21 represented a non-triple helix pyranopolysaccharide and selenium occurred in the form of C-O-Se and SeO. Molecular size and morphology studies showed that Se-POP-21 exhibited a spherical shape with a particle size distribution between 100 and 200 nm, even though Se-POP-21 aggregates were also found with a size between 500 and 600 nm. In addition, Se-POP-21 showed strong scavenging capacity to DPPH and hydroxyl radical. More, cell experiments showed that Se-POP-21 could reduce viability of A549, SKOV3, HepG2 and MCF-7 cells, induce apoptosis and inhibit metastasis of A549 cells. A potential mechanism was that Se-POP-21 inhibited the epithelial-to-mesenchymal transition of cancer cells. Se-POP-21 featured no significant effect on normal cells. Se-POP-21 showed great potential to develop into a natural antioxidant or low-toxic antitumor drug.

Glucose counteracts wood-dependent induction of lignocellulolytic enzyme secretion in monokaryon and dikaryon submerged cultures of the white-rot basidiomycete Pleurotus ostreatus.

The secretome complexity and lignocellulose degrading capacity of Pleurotus ostreatus monokaryons mkPC9 and mkPC15 and mated dikaryon dkN001 were studied in submerged liquid cultures containing wood, glucose, and wood plus glucose as carbon sources. The study revealed that this white-rot basidiomycete attacks all the components of the plant cell wall. P. ostreatus secretes a variety of glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases, especially when wood is the only carbon source. The presence of wood increased the secretome complexity, whereas glucose diminished the secretion of enzymes involved in cellulose, hemicellulose and pectin degradation. In contrast, the presence of glucose did not influence the secretion of redox enzymes or proteases, which shows the specificity of glucose on the secretion of cellulolytic enzymes. The comparison of the secretomes of monokaryons and dikaryons reveals that secretome complexity is unrelated to the nuclear composition of the strain.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of ß-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Ligno(hemi)cellulolytic Enzyme Profiles during the Developmental Cycle of the Royal Oyster Medicinal Mushroom Pleurotus eryngii (Agaricomycetes) Grown on Supplemented Agri-Wastes.

We have determined the production profiles of major ligno(hemi)cellulolytic enzymes at different stages of the mushroom development cycle during industrial scale cultivation of Pleurotus eryngii on supplemented agri-wastes. Endo-1,4-ß-glucanase, cellobiohydrolase and endoxylanase levels remained relatively low during substrate colonization, increased sharply when small fruit bodies appeared, and peaked at maturation. ß-Glucosidase and ß-xylosidase levels decreased when substrate colonization was complete, increased with the appearance of small fruit bodies and primordia, respectively, and reached maxima at maturation. Laccase peaked along with substrate colonization but, after falling sharply in the upper substrate layers, remained relatively low until postinduction. Levels increased slightly when primordia appeared, fell to minimal values during the small and mature fruit body stages, and increased again postharvest. Manganese peroxidase (Mn-P) exhibited a similar pattern initially but high enzyme levels also coincided with primordia formation. Laccase and Mn-P activity patterns were compatible with a lignin-degradation function associated with substrate colonization and, in the former case, a putative role in fruit body morphogenesis. Based on the relatively low levels of polysaccharidases recorded during the initial stages of substrate colonization, we conclude that reducing sugar levels in noncolonized substrate were adequate for sustainable vegetative growth at that stage. We further conclude that the increase in enzyme production later in the developmental cycle was consistent with the replenishment of depleted reducing sugar from cellulose in the growth substrate to levels required for fruit body formation. These data provide new information describing combined temporal and spatial enzyme production profiles throughout the mushroom development cycle under a set of conditions used in industrial scale production.

Comparison of Mono- and Dikaryotic Medicinal Mushrooms Lignocellulolytic Enzyme Activity.

Mono- and dikaryotic medicinal mushroom strains isolated from four wood-rotting basidiomycete fruiting bodies were comparatively evaluated for laccase, manganese peroxidase, cellulase, and xylanase activities in submerged cultivation in glucose or mandarin peel-containing media. Mandarin peels appeared to be the preferred growth substrate for laccase production by both mono- and dikaryotic Trametes multicolor 511 and T. versicolor 5 while glucose favored laccase activity secretion by Pleurotus ostreatus 2175. Lignocellulose-deconstructing enzyme profiles were highly variable between the studied monokaryotic and dikaryotic strains. A distinctive superiority of enzyme activity of the dikaryotic Trametes versicolor 5 and P. ostreatus 2175 over the same species monokaryotic isolates was revealed. By contrast, laccase, cellulase, and xylanase activities of the monokaryotic strain of T. multicolor 511 were rather higher than those in the dikaryotic culture. At the same time, hydrolases activity of Schizophyllum commune 632 was practically independent of the origin of the fungal culture. The results suggest that the monokaryotic isolates derived from the basidiomycetes fruiting bodies inherit parental properties but the capacity of individual monokaryotic cultures to produce lignocellulose-deconstructing enzymes can vary considerably.

Solid-State Treatment of Castor Cake Employing the Enzymatic Cocktail Produced from Pleurotus djamor Fungi.

In this work, the enzymatic cocktail produced by Pleurotus djamor fungi extracted at pH of 4.8 and 5.3 was employed for castor cake solid-state treatment. Proximal, X-ray powder diffraction and scanning electron microscopy analysis of the pristine castor cake were carried out. First, Pleurotus djamor stain was inoculated in castor cake for the enzymatic production and the enzymatic activity was determined. The maximum enzymatic activity was identified at days 14 (65.9 UI/gss) and 11 (140.3 UI/gss) for the enzymatic cocktail obtained at pH 5.3 and 4.8, respectively. Then, the enzymatic cocktail obtained at the highest enzymatic activity days was employed directly over castor cake. Lignin was degraded throughout incubation time achieving a 47 and 45% decrease for the cocktail produced at pH 4.8 and 5.3, correspondingly. These results were corroborated by the SEM and XRD analysis where a higher porosity and xylan degradation were perceived throughout the enzymatic treatment.

Laccase Fermentation of Clove Extract Increases Content of Dehydrodieugenol, Which Has Neuroprotective Activity against Glutamate Toxicity in HT22 Cells.

Enzyme fermentation is a type of food processing technique generally used to improve the biological activities of food and herbal medicines. In this study, a Syzygii Flos (clove) extract was fermented using laccase derived from Trametes versicolor (LTV). The fermented clove extract showed greater neuroprotective effects against glutamate toxicity on HT22 than the non-fermented extract did. HPLC analysis revealed that the eugenol (1) and dehydrodieugenol (2) contents had decreased and increased, respectively, after fermentation. The content of 2 peaked at 1 h after fermentation to 103.50 ± 8.20 mg/gex (not detected at zero time), while that of 1 decreased to 79.54 ± 4.77 mg/gex (185.41 ± 10.16 mg/gex at zero time). Compound 2 demonstrated promising HT22 neuroprotective properties with inhibition of Ca2+ influx, the overproduction of intracellular reactive oxygen species, and lipid peroxidation. In addition, LTV showed the best fermentation efficacy compared with laccases derived from Pleurotus ostreatus and Rhus vernicifera.

Comparative Cold Shock Expression and Characterization of Fungal Dye-Decolorizing Peroxidases.

Dye-decolorizing peroxidases (DyPs) from Auricularia auricula-judae, Bjerkandera adusta, Pleurotus ostreatus and Marasmius scorodonius (Basidiomycota) were expressed in Escherichia coli using the cold shock-inducible expression system pCOLD I DNA. Functional expression was achieved without the addition of hemin or the co-expression of any chaperones. The presence or absence of the native signal sequence had a strong impact on the success of the expression, but the effect was not consistent for the different DyPs. While BaDyP and AajDyP were stable at 50 °C, the more thermolabile MsP2 and PoDyp, upon catalytic intervention, lend themselves to more rapid thermal inactivation. The bleaching of norbixin (E 160b) using MsP2 was most efficient at pH 4.0, while BaDyP and AajDypP worked best in the weakly acidic to neutral range, indicating a choice of DyPs for a broad field of applications in different food matrices.

Secretome analysis of Pleurotus eryngii reveals enzymatic composition for ramie stalk degradation.

Pleurotus eryngii (P. eryngii) can secrete large amount of hydrolytic and oxidative enzymes to degrade lignocellulosic biomass. In spite of several researches on the individual lignolytic enzymes, a direct deconstruction of lignocellulose by enzyme mixture is not yet possible. Identifying more high-performance enzymes or enzyme complexes will lead to efficient in vitro lignocelluloses degradation. In this report, secretomic analysis was used to search for the new or interesting enzymes for lignocellulose degradation. Besides, the utilization ability of P. eryngii to ramie stalk substrate was evaluated from the degradation of cellulose, hemicellulose, and lignin in medium and six extracellular enzymes activities during different growth stages were discussed. The results showed that a high biological efficiency of 71% was obtained; cellulose, hemicelluloses, and lignin decomposition rates of P. eryngii were 29.2, 26.0, and 51.2%, respectively. Enzyme activity showed that carboxymethyl cellulase, xylanase, laccase, and peroxidase activity peaks appeared at the primordial initiation stage. In addition, we profiled a global view of the secretome of P. eryngii cultivated in ramie stalk media to understand the mechanism behind lignocellulosic biomass hydrolysis. Eighty-seven nonredundant proteins were identified and a diverse group of enzymes, including cellulases, hemicellulases, pectinase, ligninase, protease, peptidases, and phosphatase implicated in lignocellulose degradation were found. In conclusion, the information in this report will be helpful to better understand the lignocelluloses degradation mechanisms of P. eryngii.

Optimization of Arundo donax Saccharification by (Hemi)cellulolytic Enzymes from Pleurotus ostreatus.

An enzymatic mixture of cellulases and xylanases was produced by Pleurotus ostreatus using microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis of Arundo donax bioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreated A. donax. As the most significant influence during the enzymatic hydrolysis of A. donax was exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion by P. ostreatus cellulase and xylanase was analyzed by a 3(3) factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45 °C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes.

Characterization of interspecific hybrid dikaryons of the oyster mushrooms, Pleurotus florida PAU-5 and P. sajor-caju PAU-3 (higher Basidiomycetes) from India.

Five Pleurotus hybrid dikaryons, developed through cross-breeding of P. florida PAU-5 (PF-5) and P. sajor-caju PAU-3 (PSC-3) were characterized with respect to textural properties, color, and enzymatic and genetic variability. Texture profile revealed significant differences in springiness, resilience, cohesiveness, and chewiness between all hybrids compared to the parents. Among the hybrid cultures, maximum whiteness was reported in hybrid 37, whereas hybrid 8 had minimum whiteness. Three hybrids (16, 37, 42) showed an increased linear growth rate in relation to PF-5, whereas no hybrid showed a higher growth rate than PSC-3. Maximum endoglucanase and xylanase activity was observed in hybrid 46, whereas minimum activity occurred in hybrid 42. Laccase and protease activity was higher in hybrid 37 and 46, respectively. Four hybrids (16, 37, 42, 46) showed increased peroxidase activity in relation to PF-5, whereas hybrid 46 showed activity higher than the parent PSC-3. Comparison of isozyme patterns confirmed the hybrid nature of hybrid 16. The large variation in the intensity of bands could be a result of recombination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of extracellular enzymes revealed 60.3- and 43-KDa bands in all the hybrids. An additional 25-KDa band was reported in hybrids 37, 42, and 46 and the parent PF-5, indicating their close relatedness. Parental strains showed higher divergence in small-subunit ribosomal DNA region compared with the internal transcribed spacer region, indicating their significance in varietal discrimination. Hybrid 46 had a small-subunit ribosomal DNA region more similar to that of PSC-3 compared with PF-5, whereas the internal transcribed spacer region of hybrids 42 and 46 revealed close resemblance to that of PF-5 and PSC-3, respectively.

Immobilization of a Pleurotus ostreatus laccase mixture on perlite and its application to dye decolourisation.

In the present study, a crude laccase preparation from Pleurotus ostreatus was successfully immobilized on perlite, a cheap porous silica material, and tested for Remazol Brilliant Blue R (RBBR) decolourisation in a fluidized bed recycle reactor. Results showed that RBBR decolourisation is mainly due to enzyme action despite the occurrence of dye adsorption-related enzyme inhibition. Fine tuning of immobilization conditions allowed balancing the immobilization yield and the resulting rate of decolourisation, with the adsorption capacity of the solid biocatalyst. In the continuous lab scale reactor, a maximum conversion degree of 56.1% was achieved at reactor space-time of 4.2 h. Stability and catalytic parameters of the immobilized laccases were also assessed in comparison with the soluble counterparts, revealing an increase in stability, despite a reduction of the catalytic performances. Both effects are most likely ascribable to the occurrence of multipoint attachment phenomena.

Isolation and characterization of wild-type lipoxygenase LOX(Psa)1 from Pleurotus sapidus.

The lipoxygenase LOX(Psa) 1 of Pleurotus sapidus, originally investigated because of its ability to oxidize (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was isolated from the peptidase-rich lyophilisate using a three-step purification scheme including preparative isoelectric focusing and chromatographic techniques. Nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS) of the purified enzyme and peptide mass fingerprint analysis gave 38 peptides of the lipoxygenase from P. sapidus. Nearly 50% of the 643 amino acids long sequence encoded by the cDNA was covered. Both terminal peptides of the native LOX(Psa) 1 were identified by de novo sequencing, and the postulated molecular mass of 72.5 kDa was confirmed. With linoleic acid as the substrate, the LOX(Psa)1 showed a specific activity of 113 U mg(-1) and maximal activity at pH 7.0 and 30 degrees C, respectively.

Biodegradation of aflatoxin B1 in contaminated rice straw by Pleurotus ostreatus MTCC 142 and Pleurotus ostreatus GHBBF10 in the presence of metal salts and surfactants.

Aflatoxin B1 (AFB1) is a highly toxic fungal metabolite having carcinogenic, mutagenic and teratogenic effects on human and animal health. Accidental feeding of aflatoxin-contaminated rice straw may be detrimental for ruminant livestock and can lead to transmission of this toxin or its metabolites into the milk of dairy cattle. White-rot basidiomycetous fungus Pleurotus ostreatus produces ligninolytic enzymes like laccase and manganese peroxidase (MnP). These extracellular enzymes have been reported to degrade many environmentally hazardous compounds. The present study examines the ability of P. ostreatus strains to degrade AFB1 in rice straw in the presence of metal salts and surfactants. Laccase and MnP activities were determined spectrophotometrically. The efficiency of AFB1 degradation was evaluated by high performance liquid chromatography. Highest degradation was recorded for both P. ostreatus MTCC 142 (89.14 %) and P. ostreatus GHBBF10 (91.76 %) at 0.5 µg mL(-1) initial concentration of AFB1. Enhanced degradation was noted for P. ostreatus MTCC 142 in the presence of Cu(2+) and Triton X-100, at toxin concentration of 5 µg mL(-1). P. ostreatus GHBBF10 showed highest degradation in the presence of Zn(2+) and Tween 80. Liquid chromatography-mass spectrometric analysis revealed the formation of hydrated, decarbonylated and O-dealkylated products. The present findings suggested that supplementation of AFB1-contaminated rice straw by certain metal salts and surfactants can improve the enzymatic degradation of this mycotoxin by P. ostreatus strains.

Effects of different selenium levels on growth and regulation of laccase and versatile peroxidase in white-rot fungus, Pleurotus eryngii.

This study was conducted to evaluate the physiological effects of different selenium (Se) levels on the growth of white-rot fungus, Pleurotus eryngii, with special reference to the regulation of ligninolytic enzymes such as laccase and versatile peroxidase. The fungus was grown in medium supplemented with 1, 10, 100, 1,000 and 10,000 µM of sodium selenite. Mycelial growth was stronger at lower Se levels, but declined significantly at higher concentrations of 1,000 and 10,000 µM, highlighting its association in mediating toxic responses. Inhibition of fungal growth was accompanied with dense and entangled hyphae taking the shape of irregular short strips. Additionally, hyphal swellings and septation were noticed which lead to a reduction in the advancement of the mycelium. Along with the inhibition of fungal biomass, the reducing sugar and protein concentrations increased to about 30.2 and 3.5 mg/ml respectively in the growth medium. Additionally, the laccase gene expression showed a twofold upregulation at higher levels of Se, although the activity of the enzyme was compromised with an inverse relationship with increased gene transcripts. The versatile peroxidase transcript showed a complete downregulation at 10,000 µM after an upregulation at lower levels of Se. We also confirmed the direct relationship of different Se levels on laccase activity of Rhus vernicifera that showed similar behavior to the fungal laccase. The results of the present study suggest that Se supplementation regulates mRNA levels of laccase and versatile peroxidase depending on exposure and may play a role in the toxicity associated with Se.

Molecular characterization of a lipoxygenase from the basidiomycete mushroom Pleurotus ostreatus.

The full-length cDNA of the gene PoLOX1 encoding a lipoxygenase (LOX) and its corresponding genomic DNA were isolated from the basidiomycete mushroom Pleurotus ostreatus strain H1. The deduced amino acid sequence of PoLOX1 showed similarity to a valencene dioxygenase of Pleurotus sapidus, putative LOX-like proteins from ascomycete, basidiomycete, and deuteromycete fungi, and known LOXs from plants, animals, and bacteria. PoLOX1 also contained the LOX iron-binding catalytic domain in the C-terminal region, but not the polycystin-1, lipoxygenase, alpha-toxin (PLAT) domain, which is usually found in the N-terminal region of eukaryotic LOXs. Genomic sequence analysis revealed that PoLOX1 was interrupted by one intron, and that the promoter region included TATA and CAAT boxes. Southern blot analysis indicated that PoLOX1 is a member of a small gene family comprising highly similar genes. Northern blot analysis revealed that it is transcribed more abundantly in the stipes of the fruit bodies than in the caps.

Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus.

Thirty-nine white-rot fungi belonging to nine species of Agaricomycotina (Basidiomycota) were initially screened for their ability to decrease olive-mill wastewater (OMW) phenolics. Four strains of Ganoderma australe, Ganoderma carnosum, Pleurotus eryngii and Pleurotus ostreatus, were selected and further examined for key-aspects of the OMW biodegradation process. Fungal growth in OMW-containing batch cultures resulted in significant decolorization (by 40-46% and 60-65% for Ganoderma and Pleurotus spp. respectively) and reduction of phenolics (by 64-67% and 74-81% for Ganoderma and Pleurotus spp. respectively). COD decrease was less pronounced (12-29%). Cress-seeds germination increased by 30-40% when OMW was treated by Pleurotus strains. Toxicity expressed as inhibition of Aliivibrio fischeri luminescence was reduced in fungal-treated OMW samples by approximately 5-15 times compared to the control. As regards the pertinent enzyme activities, laccase and Mn-independent peroxidase were detected for Ganoderma spp. during the entire incubation period. In contrast, Pleurotus spp. did not exhibit any enzyme activities at early growth stages; instead, high laccase (five times greater than those of Ganoderma spp.) and Mn peroxidases activities were determined at the end of treatment. OMW decolorization by Ganoderma strains was strongly correlated to the reduction of phenolics, whereas P. eryngii laccase activity was correlated with the effluent's decolorization.

Release of Pleurotus ostreatus versatile-peroxidase from Mn2+ repression enhances anthropogenic and natural substrate degradation.

The versatile-peroxidase (VP) encoded by mnp4 is one of the nine members of the manganese-peroxidase (MnP) gene family that constitutes part of the ligninolytic system of the white-rot basidiomycete Pleurotus ostreatus (oyster mushroom). VP enzymes exhibit dual activity on a wide range of substrates. As Mn(2+) supplement to P. ostreatus cultures results in enhanced degradation of recalcitrant compounds and lignin, we examined the effect of Mn(2+) on the expression profile of the MnP gene family. In P. ostreatus (monokaryon PC9), mnp4 was found to be the predominantly expressed mnp in Mn(2+)-deficient media, whereas strongly repressed (to approximately 1%) in Mn(2+)-supplemented media. Accordingly, in-vitro Mn(2+)-independent activity was found to be negligible. We tested whether release of mnp4 from Mn(2+) repression alters the activity of the ligninolytic system. A transformant over-expressing mnp4 (designated OEmnp4) under the control of the ß-tubulin promoter was produced. Now, despite the presence of Mn(2+) in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as early as 4 days after inoculation. The level of expression was constant throughout 10 days of incubation (about 0.4-fold relative to ß-tubulin) and the activity was comparable to the typical activity of PC9 in Mn(2+)-deficient media. In-vivo decolorization of the azo dyes Orange II, Reactive Black 5, and Amaranth by OEmnp4 preceded that of PC9. OEmnp4 and PC9 were grown for 2 weeks under solid-state fermentation conditions on cotton stalks as a lignocellulosic substrate. [(14)C]-lignin mineralization, in-vitro dry matter digestibility, and neutral detergent fiber digestibility were found to be significantly higher (about 25%) in OEmnp4-fermented substrate, relative to PC9. We conclude that releasing Mn(2+) suppression of VP4 by over-expression of the mnp4 gene in P. ostreatus improved its ligninolytic functionality.