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Coprinopsis cinerea, a model fungus, is utilized for investigating the developmental mechanisms of basidiomycetes. The development of basidiomycetes is a highly organized process that requires coordination among genetic, environmental, and physiological factors. Oxylipins, a class of widely distributed signaling molecules, play crucial roles in fungal biology. Among oxylipins, the sexual pheromone-inducing factors (psi factors) have been identified as key regulators of the balance between asexual and sexual spore development in Ascomycetes. Linoleate dioxygenases are enzymes involved in the biosynthesis of psi factors, yet their specific physiological functions in basidiomycete development remain unclear. In this study, linoleate dioxygenases in basidiomycetes were identified and characterized. Phylogenetic analysis revealed that linoleate dioxygenases from Basidiomycota formed a distinct clade, with linoleate dioxygenases from Agaricomycetes segregating into three groups and those from Ustilaginomycetes forming a separate group. Both basidiomycete and ascomycete linoleate dioxygenases shared two characteristic domains: the N-terminal of linoleate dioxygenase domain and the C-terminal of cytochrome P450 domain. While the linoleate dioxygenase domains exhibited similarity between basidiomycetes and ascomycetes, the cytochrome P450 domains displayed high diversity in key sites. Furthermore, the gene encoding the linoleate dioxygenase Ccldo1 in C. cinerea was knocked out, resulting in a significant increase in fruiting body formation without affecting asexual conidia production. This observation suggests that secondary metabolites synthesized by CcLdo1 negatively regulate the sexual reproduction process in C. cinerea while not influencing the asexual reproductive process. This study represents the first identification of a gene involved in secondary metabolite synthesis that regulates basidiocarp development in a basidiomycete., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ageritin is a specific ribonuclease, extracted from the edible mushroom Cyclocybe aegerita (synonym Agrocybe aegerita ), which cleaves a single phosphodiester bond located within the universally conserved alpha-sarcin loop (SRL) of 23-28S rRNAs. This cleavage leads to the inhibition of protein biosynthesis, followed by cellular death through apoptosis. The structural and enzymatic properties show that Ageritin is the prototype of a novel specific ribonucleases family named 'ribotoxin-like proteins', recently found in fruiting bodies of other edible basidiomycetes mushrooms (e.g., Ostreatin from Pleurotus ostreatus , Edulitins from Boletus edulis , and Gambositin from Calocybe gambosa ). Although the putative role of this toxin, present in high amount in fruiting body (>2.5 mg per 100 g) of C. aegerita, is unknown, its antifungal and insecticidal actions strongly support a role in defense mechanisms. Thus, in this review, we focus on structural, biological, antipathogenic, and enzymatic characteristics of this ribotoxin-like protein. We also highlight its biological relevance and potential biotechnological applications in agriculture as a bio-pesticide and in biomedicine as a therapeutic and diagnostic agent.

L-Asparaginase is a therapeutically and industrially-competent enzyme, acting predominantly as an anti-neoplastic and anti-cancerous agent. The existing formulations of prokaryotic L-asparaginase are often toxic and contain L-glutaminase and urease residues, thereby increasing the purification steps. Production of L-glutaminase and urease free L-asparaginase is thus desired. In this research, bioprospecting of isolates from the less explored class Agaricomycetes was undertaken for L-asparaginase production. Plate assay (using phenol red and bromothymol blue dyes) was performed followed by estimation of L-asparaginase, L-glutaminase and urease activities by Nesslerization reaction for all the isolates. The isolate displaying the desired enzyme production was subjected to morphological, molecular identification, and phylogenetic analysis with statistical validation using Jukes-Cantor by Neighbour-joining tree of Maximum Likelihood statistical method. Among the isolates, Ganoderma australe GPC191 with significantly high zone index value (5.581 ± 0.045 at 120 h) and enzyme activity (1.57 ± 0.006 U/mL), devoid of L-glutaminase and urease activity was selected. The present study for the first-time reported G. australe as the potential source of L-glutaminase and urease-free L-asparaginase and also is one of the few studies contributing to the literature of G. australe in India. Hence, it can be postulated that it may find its future application in pharmaceutical and food industries.

A novel fibrinolytic enzyme, ACase was isolated from fruiting bodies of a mushroom, Agrocybe aegerita. ACase was purified by using ammonium sulfate precipitation, gel filtration, ion exchange and hydrophobic chromatographies to 237.12 fold with a specific activity of 1716.77 U/mg. ACase was found to be a heterodimer with molecular mass of 31.4 and 21.2 kDa by SDS-PAGE and appeared as a single band on Native-PAGE and fibrin-zymogram. The N-terminal sequence of the two subunits of ACase was AIVTQTNAPWGL (subunit 1) and SNADGNGHGTHV (subunit 2). ACase had maximal activity at 47 °C and pH 7.6. It's activity was improved by Cu 2+ , Na + , Fe 3+ , Zn 2+ , Ba 2+ , K + and Mn 2+ , but inhibited by Fe 2+ , Mg 2+ and Ca 2+ . PMSF, SBTI, aprotinine and Lys inhibited the enzyme activity, which suggested that ACase was a serine protease. ACase could degrade all three chains (α, β and γ) of fibrinogen. Moreover, the enzyme acted as both, a plasmin-like fibrinolytic enzyme and a plasminogen activator. It could hydrolyze human thrombin slightly, which indicated that the ACase could inhibit the activity of thrombin and acted as an anticoagulant to prevent thrombosis. Based on these results, ACase might act as a therapeutic agent for treating thrombosis, or as a functional food. Further investigation of the enzyme is underway., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A fibrinolytic enzyme with thrombolytic, anticoagulant activities was purified from fruiting bodies of wild-growing mushroom Boletus pseudocalopus Hongo and homogenized with a two-step procedure with a 6.11-fold increase in specific activity and 3.2% recovery. The molecular weight of the enzyme was estimated to be 63.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme was active at 40°C and pH 7, and activity was inhibited by zinc metal ion and by serine protease and trypsin inhibitors phenylmethylsulfonyl fluoride and N-α-tosyl-l-lysinyl-chloromethylketone. The enzyme displayed high specificity for Pyro-Glu-Gly-Arg-pNA. In vitro assays showed that the enzyme was able to degrade fibrin and blood clots, inhibit thrombin and activated factor X, and alter the density or structural change of fibrin clots. It could also delay activated partial thromboplastin time and prothrombin time. These results suggest that the enzyme may have characteristics of a trypsin or serine-like enzyme with fibrinolytic and thrombolytic activities and may have potential as an antithrombotic agent for blood clotting disorders.

Fungi produce several toxins active against plants, animal or humans. Among them, ribotoxins are enzymes that specifically attack ribosomes irreparably compromising protein synthesis, useful as insecticides or as anticancer agents. Here, a novel ribotoxin from the edible mushroom Pleurotus ostreatus has been purified and characterized. This ribotoxin, named Ostreatin, is a specific ribonuclease releasing α-fragment when incubated with yeast or rabbit ribosomes. Ostreatin shows IC 50 of 234 pM in rabbit reticulocyte lysate, and metal dependent endonuclease activity. Following the completion of Ostreatin primary structure, we ascertained that this toxin is homologous to Ageritin, the first ribotoxin-like protein from the basidiomycete Agrocybe aegerita, with which it shares 38.8% amino acid sequence identity. Ostreatin consists of 131 amino acid residues with an experimental molecular mass of 14,263.51 Da ([M+H + ] + ). Homology modeling revealed that Ostreatin and Ageritin share a similar fold in which the common catalytic triad is conserved. Purified Ostreatin lacks N-terminal and C-terminal peptides, which instead are present in the Ostreatin coding sequence. Such peptides are probably involved in protein sorting and for this they could be removed. Our findings confirm the presence of ribotoxin-like proteins in basidiomycetes edible mushrooms, that we propose as novel tool for biotechnological applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A large amount of agro-industrial waste is produced worldwide in various agricultural sectors and by different food industries. The disposal and burning of this waste have created major global environmental problems. Agro-industrial waste mainly consists of cellulose, hemicellulose and lignin, all of which are collectively defined as lignocellulosic materials. This waste can serve as a suitable substrate in the solid-state fermentation process involving mushrooms. Mushrooms degrade lignocellulosic substrates through lignocellulosic enzyme production and utilize the degraded products to produce their fruiting bodies. Therefore, mushroom cultivation can be considered a prominent biotechnological process for the reduction and valorization of agro-industrial waste. Such waste is generated as a result of the eco-friendly conversion of low-value by-products into new resources that can be used to produce value-added products. Here, we have produced a brief review of the current findings through an overview of recently published literature. This overview has focused on the use of agro-industrial waste as a growth substrate for mushroom cultivation and lignocellulolytic enzyme production.

Acetyl-CoA C-acetyltransferase synthase gene (AACT) cDNA, DNA and promoter were cloned from Sanghuangporus baumii. The gene ORF (1260 bp) encoded 419 amino acids. The AACT DNA includes five exons (1-84 bp, 140-513 bp, 570-1027 bp, 1090-1282 bp, 1344-1494 bp) and four introns (85-139 bp, 514-569 bp, 1028-1089 bp, 1283-1343 bp). The molecular weight of AACT protein is 43.40 kDa, it is hydrophilic with a theoretical isoelectric point of 8.96. Furthermore, The region of the transcription start site is 1997-2047 bp of AACT promoter, and it contained promoter elements (TATA Boxs, CAAT Boxs, CAAT-box, ABRE, G-Boxs, Sp1, MSA-like, LTR). AACT recombinant protein (43.40 KDa + Tag protein 22.68 KDa) was subjected in SDS-PAGE. AACT the transcription levels of in different development stages were investigated. The expression of AACT in primordia (2.4-fold) and 15 d mycelia (2.3- fold) were significantly higher than 9 d mycelia (contral). The expression level of the AACT downstream genes and triterpenoids content were determined at different developmental stages. Triterpenoid content reached its peak on day 15(7.21 mg/g)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rationale: Lys-N, also known as lysine-specific metalloendopeptidase, functions as the "sister" enzyme of lysyl endopeptidase (Lys-C) in proteomic research. Its digestion specificity at the N-terminal lysine residue makes it a very useful tool in proteomics analysis, especially in mass spectrometry (MS)-based de novo sequencing of proteins., Methods: Here we present a complete production process of highly purified Lys-N from dry fruit of Grifola frondosa (maitake mushroom). The purification process includes one step of microfiltration plus one step of UF/DF (ultrafiltration used in tandem with a diafiltration method) recovery and four steps of chromatographic purification., Results: The overall yield of the process was approximately 6.7 mg Lys-N protein/kg dry fruit of G. frondosa. The assay data demonstrated that the purified Lys-N exhibited high enzymatic activity and specificity., Conclusions: The novel production process provides for the first time the extraction of Lys-N from dry fruit of G. frondosa. The process is also stable and scalable, and provides an economic way of producing the enzyme in large quantities for MS-based proteomics and other biological studies., (© 2019 John Wiley & Sons, Ltd.)

Isogenic virus-cured and virus-infected fungal strains were previously obtained and compared to investigate mycoviral diseases and, specifically, the influence of viral infection on the vegetative growth of Pleurotus ostreatus. The present study demonstrated that infection with mycovirus PoV-ASI2792 (PoV) caused phenotypic and physiological changes in fungal cells and mycelia. The microscopically determined growth rate of the virus-infected strain was lower than that of the virus-cured strain, due to the conglomerate phenomenon during the mycelial growth process. An exploration of the viral effects of PoV on fruiting bodies yield showed significantly lower than that on virus-cured P. ostreatus. A colorimetric assay of polyphenol oxidase activity in the strains showed very weak activity in the virus-infected strain. To estimate the activity levels of enzymes related to the growth and fruiting body formation, the relative expression levels of genes encoding various extracellular enzymes such as Carbohydrate-Active Enzymes (CAZymes) were measured by quantitative RT-PCR. The expression levels of the assayed genes were significantly lower in virus-infected than in virus-cured P. ostreatus. Together, these results indicate that PoV infection affects the spawn growth and fruiting body formation of P. ostreatus via decreased expression and activity of some extracellular enzymes including lignocellulolytic enzymes.

Total phenolics, flavonoids, and polysaccharides, and individual ganoderic acid (GA) contents, antioxidant capacity, and transcription levels of key enzyme genes involved in GA biosynthesis in pileus and stipes of Ganoderma lucidum fruiting body at different growth stages were investigated in this study. Results showed that the highest total phenolics and total flavonoids contents were determined in stipes at spore maturity stage, resulting in high antioxidant activity, while the highest total polysaccharide content was found in pileus at the same stage. The pileus contained more GA than the stipes, and higher contents of ganoderic acid A and D were found at fruiting body mature stage while that of ganoderic acid B, C2, and G were found at bud elongation stage. Results from quantitative real-time PCR indicated that higher gene transcription levels of hydroxyl methylglutaryl-CoA reductase (hmgr), farnesyl pyrophosphate synthase (fps), squalene synthase (sqs), and oxidosqualene cyclase (osc) were found in pileus at bud elongation stage. Our findings will be helpful for understanding the biosynthesis of bioactive components and determining the harvest time for the desired G. lucidum fruiting bodies.

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-β-glucanase, cellobiohydrolase and endoxylanase levels remained relatively low during substrate colonization, increased sharply when small fruit bodies appeared, and peaked at maturation. β-Glucosidase and β-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.

A ribonuclease was purified from dry fruiting bodies of the wild edible mushroom Lepista personata (LPR) to 259-fold with a specific activity of 280 U/mg. The purification protocol involved ion-exchange chromatography on DEAE-cellulose, CM-cellulose and SP-sepharose, followed by size exclusion chromatography on Superdex 75. LPR is a homodimeric protein with a molecular weight of 27.8 kDa as determined by SDS-PAGE and by gel filtration. Three inner peptide sequences for LPR were obtained by LC-MS-MS analysis. It demonstrated the optimum pH of 4.0 and temperature optimum of 60°C. The specificity ribonuclease potencies order toward polyhomoribonucleotides was poly C > poly A > poly G > poly U. The ribonuclease inhibited HIV-1 reverse transcriptase with an IC50 of 0.53 μM.

Background: Mushrooms have been widely considered as health foods as their extracts have anti-hypertensive and anti-tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in this study, proteins extracted from reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin-converting enzyme (ACE) inhibitory activities were investigated., Results: Various glycoside hydrolases (GHs), such as β-N-acetylhexosaminidase (GH family 20), α-1,2-mannosidase (GH family 47), endo-β-1,3-glucanase (GH family 128), and β-1,3-glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated β-oligosaccharides. Additionally, the glutamic acid protease of the peptidase G1 family was determined as the major protein in the extract, and the residual peptidase activity of the extracts was found to improve ACE inhibitory activities. Finally, it was observed that extraction at 50 °C is suitable for yielding functional molecules with high ACE inhibitory activities., Conclusion: Water extraction is generally believed to extract only functional macromolecules that exist in mushroom fruiting bodies. This study proposed a new concept that describes how functional molecules are produced by enzymes, including proteases and GHs, during extraction. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

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.

A novel acidic α-galactosidase (EC 3.2.1.22) designated as Leucopaxillus tricolor α-galactosidase (LTG) has been purified to homogeneity from the fruiting bodies of L. tricolor to 855-fold with a specific activity of 956 U mg -1 by the application of chromatography and gel filtration. The molecular mass of LTG was estimated to be 60 kDa as determined by both SDS-PAGE and by gel filtration. The purified enzyme was identified by LC-MS/MS and four inner amino acid sequences were obtained. When 4-nitrophenyl α-D-glucopyranoside (pNPGal) was used as substrate, the optimal pH and optimal temperature of LTG were pH 5.0 and 50 °C, respectively. The enzyme activity was strongly inhibited by Hg 2+ , Fe 3 , Cu 2+ , Cd 2+ , and Mn 2+ ions. The chemical modification agent N-bromosuccinimide (NBS) completely inhibited the enzyme activity of LTG, indicating the paramount importance of tryptophan residue(s) to its enzymatic activity. Besides, LTG displayed wide substrate diversity with activity toward a variety of substrates such as stachyose, raffinose, melibiose, locust bean gum, and guar gum. Given the good ability of degrading the non-digestible and flatulence-causing oligosaccharides, this fungus may become a useful source of α-galactosidase for multiple applications., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fruiting bodies of Ganoderma lucidum have been widely used as a source of potent nutraceutical products. However, the key proteins involved in fructifying G. lucidum, to our knowledge, have not yet been reported. We evaluated the protein profile of fruiting and nonfruiting G. lucidum strains at various developmental stages: mycelia, spawn running, pinning, and fruiting body. Four strains of G. lucidum (GL-I to GL-IV) were grown in both liquid medium (mushroom minimal medium broth) and bags of wheat straw, after which the biomass and fruiting bodies were harvested. Enzyme studies revealed enhanced intracellular and extracellular enzymatic activities during the spawn run stage compared with that during mycelial growth in broth. The esterase and peroxidase activities increased significantly during the pinning of the fruiting cultures, thus indicating their positive role in fructification. Fourier transform infrared spectroscopy of proteins at 3 stages of cultivation-spawn run, pin head formation, and fruiting-exhibited the presence of hydrophobic amino acids and an ordered protein structure in fruiting strains (GL-I and GL-II), indicating the presence of hydrophobin proteins and their role in mushroom fructification. However, basic and aromatic amino acids predominated in the nonfruiting strain GL-IV, and an unordered protein structure was present, which indicate the positive role of hydrophobic amino acids and hydrophobin proteins in mushroom fructification.

A novel protease was isolated from Coprinopsis atramentaria, which is, to our knowledge, the first macromolecule to be purified from this species. The protease, referred to as CAP, was purified through the use of ion exchange chromatography on sulphopropyl-sepharose, diethylaminoethyl-cellulose, Q-Sepharose, and gel filtration on a Superdex 75 column. CAP is a monomelic protein with a molecular mass of 32 kDa. The maximum activity of CAP was detected at pH 7.0 and 50°C. The preferred pH of CAP demonstrated that it was a neutral protease. Kinetic constants were determined under optimal reaction conditions, using 1% casein as the substrate. We found Km and Vmax values of 7.98 mg · mL-1 and 215 μg · mL-1 · min-1, respectively. Among the various metal ions tested, Pb2+, Zn2+, Mn2+, Hg2+, Cu2+, and Cd2+ exerted dose-dependent inhibitory actions, whereas Mg2+ exhibited a promoting action at all concentrations tested. Eight inner peptide sequences were detected by liquid chromatography on an LTQ-Orbitrap mass spectrometer and were identified using the Basic Local Alignment Search Tool, which contains proteases from other sources. Alignment results showed that 2 of them were homologous to fungal cysteine proteases. The other 5 peptide sequences also shared similarities with proteases of other origins. The isolation of a novel protease from C. atramentaria in this study not only expands the sources of proteases but also provides further information about this fungus.

A fibrinolytic metalloprotease with in vitro fibrinolytic effects was purified from the edible mushroom Pleurotus ferulae using several chromatography steps including anion and ion exchange, gel filtration, and fast protein liquid chromatography columns. The molecular mass of the enzyme was estimated to be 20.0 kDa, as determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin zymography. The protease was active at 50°C, and pH 4.0, 5.0, and 8.0. The fibrinolytic activity of the enzyme was inhibited by ethyleneglycol-bis-(2-aminoethyl)-N,N,N',N' tetraacetic acid and strongly inhibited by two metal ions, Cu and Mg. In vitro assays evaluating fibrinolytic activity on a fibrin plate, fibrin turbidity, and thrombolytic activity on fibrin clots using human fibrinogen and human thrombin revealed that the enzyme could hydrolyze fibrin polymers directly and inhibit the formation of fibrin clots. In activated partial thromboplastin time (APTT) and prothrombin time assays, the enzyme strongly prolonged the APTT, which detects an activity of intrinsic and common pathways. The enzyme showed strong in vivo protective effect against mortality/paralysis from epinephrine plus collagen-induced acute thromboembolism in in vivo model. Our findings suggest that the enzyme may have a potential for treatment and prevention of thrombosis-relative diseases.

In this study, a 39-kDa metalloprotease was purified from a rare edible mushroom with health-promoting activities, Oudemansiella radicata, using a purification protocol which entailed anion exchange chromatography on DEAE-cellulose and Q-Sepharose columns and gel filtration by fast protein liquid chromatography on a Superdex 75 column. Some peptide sequences were obtained by LC-MS/MS analysis and one of the sequences, DAWIQADVNR, manifested 90% identity to Coprinopsis cinerea metalloprotease. The optimal reaction pH and temperature for Oudemansiella radicata protease were pH 7.0 and 50°C, respectively. The protease was purified 79-fold and demonstrated a specific protease activity of 2.42 U/mg. The K m of the purified protease for the casein substrate was 0.65 mg/mL at pH 7.0 and 50°C. The activity of the protease was inhibited by Cd 2+ , Hg 2+ , Cu 2+ , Pb 2+ and Fe 3+ ions, but was enhanced by K + , Mn 2+ and Fe 2+ ions. The marked suppression of the protease activity by EDTA indicates that the protease is a metalloprotease.

The feasible use of aqueous two-phase systems (ATPSs) to establish a viable protocol for the recovery of laccase from processed Hericium erinaceus (Bull.:Fr.) Pers. fruiting bodies was evaluated. Cold-stored (4.00±1.00°C) H. erinaceus recorded the highest laccase activities of 2.02±0.04 U/mL among all the processed techniques. The evaluation was carried out in twenty-five ATPSs, which composed of polyethylene glycol (PEG) with various molecular weights and potassium phosphate salt solution to purify the protein from H. erinaceus. Optimum recovery condition was observed in the ATPS which contained 17% (w/w) PEG with a molecular weight of 8000 and 12.2% (w/w) potassium phosphate solution, at a volume ratio (VR) of 1.0. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 99% with a purification factor of 8.03±0.46. The molecular mass of laccases purified from the bottom phase was in the range of 55-66 kDa. The purity of the partitioned laccase was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Unlabelled: The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general., Importance: The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood. The first fungal STRIPAK was described in Sordaria macrospora, which is a well-established model organism used to study the formation of fungal fruiting bodies, three-dimensional organ-like structures. We analyzed STRIPAK subunit PP2Ac1, catalytic subunit 1 of protein phosphatase PP2A, to study the importance of the catalytic activity of this protein during sexual development. The results of our yeast two-hybrid analysis and tandem affinity purification, followed by mass spectrometry, indicate that PP2Ac1 activity connects STRIPAK with other signaling pathways and thus forms a large interconnected signaling network., (Copyright © 2016 Beier et al.)

As the first defence for cells to counteract the toxicity of active oxygen, superoxide dismutase (SOD) plays an important role in the response of living organisms to stress and cell differentiation. One extracellular Cu-ZnSOD (ecCu-ZnSOD), and two MnSODs, were identified based on the Volvariella volvacea genome sequence. All three genes have complicated alternative splicing modes during transcription; only when the fourth intron is retained can the Vv_Cu-Znsod1 gene be translated into a protein sequence with SOD functional domains. The expression levels of the three sod genes in the pilei are higher than in the stipe. The Vv_Cu-Znsod1 and the Vv_Mnsod2 are co-expressed in different developmental stages of the fruiting body, with the highest level of expression in the pilei of the egg stage, and they show a significant, positive correlation with the efficiency of karyogamy, indicating the potential role of these two genes during karyogamy. The expression of the ecCu-Znsod and two Vv_Mnsod genes showed a significant up-regulated when treated by cold stress for one hour; however, the lack of the intracellular Cu-ZnSOD encoding gene (icCu-Znsod) and the special locus of the ecCu-Znsod gene initiation codon suggested a possible reason for the autolysis phenomenon of V. volvacea in cold conditions.

A novel laccase was purified from the white rot fungus, Hohenbuehelia serotina, to investigate the applications of this laccase in the decoloration of various dyes. SDS-PAGE revealed a single band of this laccase corresponding to a molecular weight of approximately 57.8 kDa. The enzyme showed activity towards several substrates, the most sensitive of which was 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). The highest enzymatic activity using ABTS as a substrate was observed at pH 6.8 and 30°C. The enzyme activity was found to be significantly enhanced in the presence of Zn(2+) ions and inhibited by Fe(2+) ions. Moreover, SDS and β-mercaptoethanol were inhibitory, and inhibition by L-cysteine was observed while EDTA and DMSO had almost no inhibitory effect. The laccase could effectively decolorize seven different dyes within 30 minutes at 40°C.

Autolysis of Coprinopsis cinerea fruiting bodies affects its commercial value. In this study, a mutant of C. cinerea that exhibits pileus expansion without pileus autolysis was obtained using ultraviolet mutagenesis. This suggests that pileus expansion and pileus autolysis involve different enzymes or proteins. Among the detected hydrolytic enzymes, only β-1,3-glucanase activity increased with expansion and autolysis of pilei in the wild-type strain, but the increase was abolished in the mutant. This suggests that β-1,3-glucanases plays a major role in the autolysis. Although there are 43 possible β-1,3-glucoside hydrolases genes, only 4 known genes, which have products that are thought to act synergistically to degrade the β-1,3-glucan backbone of cell walls during fruiting body autolysis, and an unreported gene were upregulated during pileus expansion and autolysis in the wild-type stain but were suppressed in the mutant. This suggests that expression of these β-1,3-glucanases is potentially controlled by a single regulatory mechanism.

Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

Using a combined chromatography method, we simultaneously purified three protein fractions (II-2, II-3 and II-4) with 1,3-β-glucanase activity from extraction of pilei of Coprinopsis cinerea fruiting bodies. MALDI-TOF/TOF amino acid sequencing showed that these three fractions matched a putative exo-1,3-β-glucanase, a putative glucan 1,3-β-glucosidase and a putative glycosyl hydrolase family 16 protein annotated in the C. cinerea genome, respectively; however, they were characterized as a 1,3-β-glucosidase, an exo-1,3-β-glucanase and an endo-1,3-β-glucanase, respectively, by analysis of their substrate specificities and modes of action. This study explored how these three 1,3-β-glucoside hydrolases synergistically acted on laminarin: the endo-1,3-β-glucanase hydrolysed internal glycosidic bonds of laminarin to generate 1,3-β-oligosaccharides of various lengths, the exo-1,3-β-glucanase cleaved the longer-chain laminarioligosaccharides into short-chain disaccharides, laminaribiose and gentiobiose, and the 1,3-β-glucosidase further hydrolysed laminaribiose to glucose. The remaining gentiobiose must be hydrolysed by other 1,6-β-glucosidases. Therefore, the endo-1,3-β-glucanase, exo-1,3-β-glucanase and 1,3-β-glucosidase may act synergistically to completely degrade the 1,3-β-glucan backbone of the C. cinerea cell wall during fruiting body autolysis. These three 1,3-β-glucoside hydrolases share a similar optimum pH and optimum temperature, supporting the speculation that these enzymes work together under the same conditions to degrade 1,3-β-glucan in the C. cinerea cell wall during fruiting body autolysis.

Tricholoma matsutake is an ectomycorrhizal fungus that dominates the microbial communities in the soil of pine and spruce forests. The mycorrhizas of this fungus have antimicrobial activity, although factors responsible for the antimicrobial activity have not been fully elucidated. The present study shows that fruit bodies of T. matsutake secreted hydrogen peroxide (H2O2), which was produced by pyranose oxidase, and that the H2O2 thus secreted strongly inhibited the growth of mycelia of the phytopathological fungus Rhizoctonia solani. These findings suggest that fruit bodies of T. matsutake have antifungal activity and that the pyranose oxidase plays an important role in the antifungal activity.

Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr.) fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6). Three isoforms of polyphenol oxidase (PPO) were discovered and two isoforms (1-l and 1-2) were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6). Specfic activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule ofthe enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 °C, and at +70 °C the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content ofthe milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m- and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol) and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

A purification protocol that encompassed anion exchange chromatography (EC) on DEAE-cellulose, cation EC on CM-cellulose, anion EC on Q-Sepharose, and fast protein liquid chromatography-gel filtration of Superdex 75 was used to isolate a ribonuclease from dried fruiting bodies of Ramaria formosa. The ribonuclease was unadsorbed on CM-cellulose but adsorbed on both DEAE-cellulose and Q-Sepharose. It displayed a molecular mass of 29-kDa in both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ranking of its ribonucleolytic activity toward polyhomoribonucleotides was poly(U) > poly(C) > poly(G) > poly(A). It exhibited a pH optimum of pH 5 and a temperature optimum at 60 °C. The ribonuclease inhibited HIV-1 reverse transcriptase with an IC50 of 3 µM., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oudemansiella radicata has been found to have ability to tolerate and accumulate heavy metals. In this study, to know about the metal tolerance and detoxification strategy of O. radicata, the tolerance responses in both cap and stipe of the fruiting body, including the copper content, the changes of thiol compounds production and antioxidant enzymes activities, caused by various copper stress (150-600 mg kg(-1)) during 2-6 days were investigated. Results showed that Cu content in the fruiting bodies increased with the increasing Cu concentrations and growing time, which was higher in cap than that in stipe. For thiols contents, the maximum level was in the sample at 300 mg kg(-1) Cu after 2 d both in cap and stipe, in accordance with superoxide dismutase (SOD) and glutathione reductase (GR) activities. Guaicol peroxidase (POD) activities reached maximum at 150 mg kg(-1) Cu after 4 d and 6 d, respectively in cap and stipe, while the maximum of catalase (CAT) activities was recorded at 300 and 600 mg kg(-1) Cu after 4 d in the cap and stipe, respectively. As a whole, low concentration of Cu stimulated the production of thiols and activated the antioxidant enzymes activities in the fruiting body of O. radicata after 2/4 d, while high-level Cu decreased the thiols production and enzymes activities after 4/6 d. Furthermore, the cap was more sensitive than the stipe to Cu exposure. Different indicators showed different responses to copper accumulation and the different fruiting part (cap and stipe) of O. radicata had ability to response the oxidative stress caused by Cu. Considering the metal accumulation and its own detoxification with short growing time, mushroom might have the potential to be used as bio-accumulator to deal with Cu exposure in the Cu-contaminated farmland soil., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The knowledge about biological activities of constituents from medicinal mushrooms belonging to the genus Tricholoma is limited. A 59-kDa laccase has now been purified from fresh fruiting bodies of the mushroom Tricholoma matsutake. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, affinity chromatography on ConA-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Of the various affinity and ion exchange chromatographic media employed, the laccase bound only on Con A-Sepharose. The activity of the laccase did not undergo major changes over the temperature range 20-80°C. However, all activity vanished following exposure to 100°C for 10 minutes. The enzyme activity varied only slightly over the pH range 3-5, with the optimal pH of 5, but exhibited a precipitous decline when the pH was increased to 6, and was undetectable at pH 8 and 9. The laccase showed activity in the decolorization of azo dyes without a mediator. Its N-terminal sequence demonstrated only slight resemblance to those of other mushroom laccases. The newly described laccase is distinctive from the previously isolated Tricholoma mushroom laccases in a number of aspects.

Lentinan from Lentinula edodes fruiting bodies (shiitake mushrooms) is a valuable β-glucan for medical purposes based on its anticancer activity and immunomodulating activity. However, lentinan content in fruiting bodies decreases after harvesting and storage due to an increase in glucanase activity. In this study, we downregulated the expression of an exo-β-1,3-glucanase, exg2, in L. edodes using RNA interference. In the wild-type strain, β-1,3-glucanase activity in fruiting bodies remarkably increased after harvesting, and 41.7% of the lentinan content was lost after 4 days of preservation. The EXG2 downregulated strain showed significantly lower lentinan degrading activity (60-70% of the wild-type strain) in the fruiting bodies 2-4 days after harvesting. The lentinan content of fresh fruiting bodies was similar in the wild-type and EXG2 downregulated strains, but in the downregulated strain, only 25.4% of the lentinan was lost after 4 days, indicating that downregulation of EXG2 enables keeping the lentinan content high longer.

Morels are wild edible ascomycetous mushrooms that are highly prized because of their medicinal and nutritional qualities. Ligninolytic enzymes are considered as one of the most important enzymes in fungi due to their involvement in fruiting body formation during artificial cultivation on different substrates. In the life cycle of morels, sclerotia are the intermediate stage to form a fruiting body from mycelia. We have studied the production of ligninolytic enzymes by Morchella crassipes MR8 growing on different substrates and during sclerotia formation. This fungus is able to produce ligninolytic enzymes such as laccase (Lac), lignin peroxidase (LiP), and manganese-dependent peroxidase (MnP) when grown on different substrates. Maximum Lac activity was observed when grown in wheat grains whereas maximum activities of MnP and LiP were observed when grown in rice straw. Laccase enzyme was produced in high titers during sclerotia formation and maturation when grown in combinations of soil and substrates. A large number of sclerotia was observed in soil and wheat grains, along with high titers of laccase. Cellulase activity was observed to be constant during sclerotia formation and maturation. The present study results suggest that laccase enzyme might play an important role in sclerotia formation in morels., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple and efficient procedure for preparation and immobilization of tyrosinase enzyme was developed utilizing whole cells from the edible mushroom Agaricus bisporus, without the need for enzyme purification. Tyrosinase activity in the cell preparation remained constant during storage at 21 °C for at least six months. The cells were entrapped in chitosan and alginate matrix capsules and characterized with respect to their resulting tyrosinase activity. A modification of the alginate with colloidal silica enhanced the activity due to retention of both cells and tyrosinase from fractured cells, which otherwise leached from matrix capsules. The observed activity was similar to the activity that was obtained with immobilized isolated tyrosinase in the same material. Mushroom cells in water were susceptible to rapid inactivation, whereas the immobilized cells maintained 73% of their initial activity after 30 days of storage in water. Application in repeated batch experiments resulted in almost 100% conversion of endocrine disrupting bisphenol A (BPA) for 11 days, under stirring conditions, and 50-60% conversion after 20 days, without stirring under continuous usage. The results represent the longest yet reported application of immobilized tyrosinase for degradation of BPA in environmental water samples., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ganoderma lucidum has drawn worldwide interest with regard to its secondary metabolism and pharmaceutical activity. However, the development of such research has been limited because of a lack of basic biological knowledge. Nicotinamide adenine dinucleotide phosphate oxidases (Nox) have recently been highlighted because of the many important biological roles in plants and animals; however, the exact functions of Nox are still not fully understood in fungi. In this study, we identified two Nox isoforms (NoxA and NoxB) and a regulator, NoxR. RNA interference was used, and silencing of the Nox isoforms and NoxR expression indicated a central role for these genes in hyphal branching, fruiting body development, reactive oxygen species (ROS) generation, ROS resistance and ganoderic acid biosynthesis regulation. Further mechanistic investigation revealed that Nox-generated ROS elevated cytosolic Ca(2+) levels by activating a plasma membrane Ca(2+) influx pathway, thereby inducing the Ca(2+) signal pathway to regulate ganoderic acid biosynthesis and hyphal branching. Importantly, our results highlight the Nox functions in signal crosstalk between ROS and Ca(2+), and these findings provide an excellent opportunity to identify the potential pathway linking ROS networks to calcium signalling in fungi and suggest that plants, animals and fungi share a conserved signal-crosstalk mechanism., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A monomeric acid phosphatase (ACP) with a molecular mass of 72.5 kDa was purified from fresh fruiting bodies of cultured Schizophyllum commune mushroom. The isolation procedure entailed ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. It demonstrated a unique N-terminal amino acid sequence of NAPWAQIDEV, which exhibited 60% amino acid identity to that of S. commune hypothetical histidine ACP based on its genome sequence, but less than 30% amino acid identity to that of other fungal ACPs previously reported. The ACP exhibited an optimum temperature at 50 °C, an optimum pH at pH 4.6, and was considerably stable at a pH range of 4.0 to 9.0, and a temperature range of 20-40 °C. The Km of the purified enzyme for ρ-nitrophenyl phosphate (ρNPP) was 0.248 mM and the Vmax was 9.093 × 10(-3)  μM/min. ACP activity was strongly inhibited by Al(3+) and Fe(3+) , but enhanced by Co(2+) , Mg(2+) , and Ca(2+) at a concentration of 0.5 mM., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The edible straw mushroom Volvariella volvacea is an important crop in South East Asia and is predominantly harvested in the egg stage. Rapid stipe elongation and cap expansion result in a swift transition from the egg to elongation and maturation stage, which are subjected to fast senescence and deterioration. In other mushrooms, β-1,3-glucanases have been associated with degradation (softening) of the cell wall during stipe elongation and senescence. We present a new glycoside hydrolase family 55 (GH55) exo-β-1,3-glucanase gene, exg2, and highly conserved deduced EXG2 protein. The 3D model and presumed catalytic residues of V. volvacea EXG2 are identical to Lentinula edodes EXG2 and Phanerochaete chrysosporium Lam55A, supporting similar enzymatic functions. In addition to previous association to stipe elongation and senescence, our data clearly indicates a role for cap (pileus) expansion. Digital gene expression, quantitative PCR and isobaric tags for relative and absolute quantification analysis showed low exg2 and EXG2 levels in primordia, button, egg and elongation stages and significantly increased levels in the maturation stage. Subsequent relative quantitative PCR analysis designated expression of exg2 to the stipe in the elongation stage and to the pileus and stipe in the maturation stage. EXG2 cell wall softening activity, close correlation of exg2 expression with the principal expanding mushroom tissues and a strong conservation of expression patterns and protein sequences in other mushrooms, make V. volvacea exg2 an important candidate for future studies on mechanisms of fruiting body expansion and senescence causing commodity value loss., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A 28-kDa ribonuclease, with an optimum pH of 4.0 and an optimum temperature at 58 °C, was isolated from fruiting bodies of the edible mushroom Hygrophorus russula. It was purified by ion exchange chromatography on carboxymethyl-cellulose, dithyaminoethyl-cellulose, quaternary amine-sepharose and sulphopropyl-sepharose, followed by fast protein liquid chromatography gel filtration on Superdex 75. The N-terminal amino acid sequence was ASAGG which showed homology to those of other fungal RNases to some degree. It exerted the highest RNase activity on poly C and poly U. The Michaelis constant (K(m)) value of the RNase on yeast tRNA was 3.6 μM, and the maximal velocity (V(max)) was 0.6 μM. The RNase activity was suppressed by some ions including Fe(2+) and Zn(2+) ions. The RNase inhibited the activity of HIV-1 reverse transcriptase with an IC(50) of 4.64 μM.

This is the first report on using Catathelasma ventricosum for production of fruiting body and lignocellulosic enzymes. To improve the laccase activity and productivity of mushroom, the substrate was added with different supplements (eight aromatic compounds, Mn2+, and Cu2+). Based on the results, all these supplements can improve the laccase activity and productivity of C. ventricosum, and it seems that there is a critical value of laccase activity that affects the productivity of C. ventricosum. In addition, when Penicillium decumbens was inoculated into the substrate that had been cultivated with C. ventricosum for 20 days, the highest values of laccase activity, FPA activity, and productivity of C. ventricosum were obtained. Moreover, the laccase activity showed a positive correlation with the productivity of C. ventricosum. Finally, the effect of Mn2+, Cu2+, and P. decumbens on laccase activity was investigated by response surface methodology (RSM).

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.

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40 degrees C. The enzyme activity was enhanced by Mn2+ and Fe3+ ions, but inhibited by Hg2+ ions.

Tricholoma matsutake is an economically important ectomycorrhizal fungus of coniferous woodlands. Mycologists suspect that this fungus is also capable of saprotrophic feeding. In order to evaluate this hypothesis, enzyme and chemical assays were performed in the field and laboratory. From a natural population of T. matsutake in southern Finland, samples of soil-mycelium aggregate (shiro) were taken from sites of sporocarp formation and nearby control (PCR-negative) spots. Soil organic carbon and activity rates of hemicellulolytic enzymes were measured. The productivity of T. matsutake was related to the amount of utilizable organic carbon in the shiro, where the activity of xylosidase was significantly higher than in the control sample. In the laboratory, sterile pieces of bark from the roots of Scots pine were inoculated with T. matsutake and the activity rates of two hemicellulolytic enzymes (xylosidase and glucuronidase) were assayed. Furthermore, a liquid culture system showed how T. matsutake can utilize hemicellulose as its sole carbon source. Results linked and quantified the general relationship between enzymes secreted by T. matsutake and the degradation of hemicellulose. Our findings suggest that T. matsutake lives mainly as an ectomycorrhizal symbiont but can also feed as a saprotroph. A flexible trophic ecology confers T. matsutake with a clear advantage in a heterogeneous environment and during sporocarp formation.

Rhinocladiella aquaspersa is an etiologic agent of chromoblastomycosis, a subcutaneous chronic infectious disease. In the present work, we found that the three morphological forms of this fungus (conidia, mycelia and sclerotic bodies) expressed different levels of ecto-phosphatase activity. Our results demonstrated that surface conidial enzyme is an acid phosphatase, inhibited by sodium salts of molybdate, orthovanadate and fluoride and that the inhibition caused by orthovanadate and molybdate was irreversible. The conidial ecto-phosphatase efficiently released phosphate groups from different phosphorylated substrates, causing a higher rate of phosphate removal when p-nitrophenylphosphate was used as substrate. This ecto-enzyme of R. aquaspersa is modulated by Co(2 +) ions and inorganic phosphate (Pi). Accordingly, removal of Pi from the culture medium resulted in a marked (121-fold) increase of ecto-phosphatase activity. Surface phosphatase activity is apparently involved in fungal adhesive properties, since the attachment of R. aquaspersa to epithelial cells was reversed by the pre-treatment of the conidia with orthovanadate, molybdate and anti-phosphatase antibody. Corroborating this finding, conidia with greater ecto-phosphatase activity (grown in Pi-depleted medium) showed higher adherence to epithelial cells than fungi cultivated in the presence of Pi.

This study focused on the tolerance responses of the fruiting body of Oudemansiella radicata towards different concentrations of lead (250-1000 mg kg(-1)) for 2-6 d. To know about the lead tolerance and detoxification strategy, the lead content, thiol content and the activities of antioxidant enzymes were investigated. The maximum level for the lead concentration in O. radicata was recorded in the 6 d sample in each treatment, and for thiols, it was recorded in the 500 mg kg(-1) Pb/2d sample, while for superoxide dismutases (SOD) and catalases (CAT) activities, it was reached at 1000 mg kg(-1) Pb after 2 d in the stipe and cap, respectively. Peroxidases (POD) activities showed a more complex trend and glutathione reductases (GR) reached the maximum at 500 mg kg(-1) Pb after 2 d in the stipe. Overall, the results showed that low concentration lead stimulated the fruiting body of O. radicata to produce the thiols and activate the antioxidant enzymes after 2 d/4 d, while high concentration Pb resulted in the decline/decrease of the thiols and the activities of antioxidant enzymes after 4 d/6 d. Benefiting from the metal accumulation, detoxification potential and the short lifetime, mushroom have the potential for bioaccumulation of heavy metal in polluted farmland., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Unlabelled: γ-Glutamyl-transpeptidase (GGT) is one of the important enzymes in the pathway of odor formation in shiitake mushroom (Lentinus edodes). Rapid purification and characterization of GGT from shiitake mushroom were studied in this work. The GGT was purified 179-fold after 3 primary steps: precipitation by ammonium sulfate, isolation by Phenyl Sepharose 6 FF, and desalting by Sephadex G-25. The enzyme, consisting of a small and a large subunit with Mr 28 KDa and 60 KDa, respectively, is composed of 17 kinds of amino acids with the ratio of basic and acidic residues 1: 1.84, and its secondary structure was also determined by Fourier transform infrared spectroscopy. The properties of GGT were studied with γ-glutamyl-p-nitroanilide as the substrate. The results showed Km value of 2.601μM, optimal temperature of 40 °C, and isoelectric point of 6.4. In addition, the activity of GGT was promoted by Na⁺, K⁺, and Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Zn²⁺, and Fe³⁺., Practical Application: In this work, the γ-glutamyl-transpeptidase (GGT) from shiitake mushroom was purified with a simple scheme. Through the characterization of GGT, the relationship between endogenous formaldehyde and odor formation is to be clarified and assist in finding means of formaldehyde control in shiitake mushroom., (© 2012 Institute of Food Technologists®)

To investigate the effects of selenium on the main active components of Cordyceps militaris fruit bodies, selenium-enriched cultivation of C. militaris and the main active components of the fruit bodies were studied. Superoxide dismutase (SOD) activity and contents of cordycepin, cordycepic acid, and organic selenium of fruit bodies were sodium selenite concentration dependent; contents of adenosine and cordycep polysaccharides were significantly enhanced by adding sodium selenite in the substrates, but not proportional to sodium selenite concentrations. In the cultivation of wheat substrate added with 18.0 ppm sodium selenite, SOD activity and contents of cordycepin, cordycepic acid, adenosine, cordycep polysaccharides, and total amino acids were enhanced by 121/145%, 124/74%, 325/520%, 130/284%, 121/145%, and 157/554%, respectively, compared to NS (non-selenium-cultivated) fruit bodies and wild Cordyceps sinensis; organic selenium contents of fruit bodies reached 6.49 mg/100 g. So selenium-enriched cultivation may be a potential way to produce more valuable medicinal food as a substitute for wild C. sinensis.

A 14.6-kDa RNase, with a pH optimum of 5.5 and a temperature optimum of 70 °C, was isolated from dried fruiting bodies of the edible mushroom Lactarius flavidulus. The purification procedure involved, in succession, ion exchange chromatography on DEAE-cellulose, CM-cellulose and SP-Sepharose, and finally FPLC-gel filtration on Superdex 75. The RNase was adsorbed on all three ion exchangers. The ranking of its activity toward various polyhomoribonucleotides was poly(C) > poly(G) > poly(A) > poly(U). It suppressed proliferation of HepG2 cells and L1210 cells with an IC₅₀ of 3.19 μM and 6.52 μM, respectively. It also inhibited the activity of HIV-1 reverse transcriptase with an IC₅₀ of 2.55 μM.

The cyclic peptide toxins of poisonous Amanita mushrooms are chemically unique among known natural products. Furthermore, they differ from other fungal cyclic peptides in being synthesized on ribosomes instead of by nonribosomal peptide synthetases. Because of their novel structures and biogenic origins, elucidation of the biosynthetic pathway of the Amanita cyclic peptides presents both challenges and opportunities. In particular, a full understanding of the pathway should lead to the ability to direct synthesis of a large number of novel cyclic peptides based on the Amanita toxin scaffold by genetic engineering of the encoding genes. Here, we highlight some of the principal methods for working with the Amanita cyclic peptides and the known steps in their biosynthesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession), and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6-8 and in the temperature range 35-60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC₅₀ value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.