Characterization of linoleate dioxygenases in basidiomycetes and the functional role of CcLdo1 in regulating fruiting body development in Coprinopsis cinerea.

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.

Ageritin from Pioppino Mushroom: The Prototype of Ribotoxin-Like Proteins, a Novel Family of Specific Ribonucleases in Edible Mushrooms.

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.

Fibrinolytic and Thrombolytic Effects of an Enzyme Purified from the Fruiting Bodies of Boletus pseudocalopus (Agaricomycetes) from Korea.

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.

Bioprospecting of the agaricomycete Ganoderma australe GPC191 as novel source for L-asparaginase production.

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 serine protease with anticoagulant and fibrinolytic activities from the fruiting bodies of mushroom Agrocybe aegerita.

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 Cu2+, Na+, Fe3+, Zn2+, Ba2+, K+ and Mn2+, but inhibited by Fe2+, Mg2+ and Ca2+. PMSF, SBTI, aprotinine and Lys inhibited the enzyme activity, which suggested that ACase was a serine protease. ACase could degrade all three chains (α, ß 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.

The ribotoxin-like protein Ostreatin from Pleurotus ostreatus fruiting bodies: Confirmation of a novel ribonuclease family expressed in basidiomycetes.

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 IC50 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.

Comparative Studies on Bioactive Compounds, Ganoderic Acid Biosynthesis, and Antioxidant Activity of Pileus and Stipes of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes) Fruiting Body at Different Growth Stages.

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.

Cultivation of Mushrooms and Their Lignocellulolytic Enzyme Production Through the Utilization of Agro-Industrial Waste.

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.

Molecular cloning, characterization, and heterologous expression of an acetyl-CoA acetyl transferase gene from Sanghuangporus baumii.

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).

The PoV mycovirus affects extracellular enzyme expression and fruiting body yield in the oyster mushroom, Pleurotus ostreatus.

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.

A Novel Ribonuclease with HIV-1 Reverse Transcriptase Inhibitory Activity Purified from the Field Blewit Mushroom Lepista personata (Agaricomycetes).

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.

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.

Peptidyl-Lys metalloendopeptidase (Lys-N) purified from dry fruit of Grifola frondosa demonstrates "mirror"digestion property with lysyl endopeptidase (Lys-C).

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.

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.

Protein components of water extracts from fruiting bodies of the reishi mushroom Ganoderma lucidum contribute to the production of functional molecules.

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 ß-N-acetylhexosaminidase (GH family 20), α-1,2-mannosidase (GH family 47), endo-ß-1,3-glucanase (GH family 128), and ß-1,3-glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated ß-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.

Hydrolysis of oligosaccharides by a fungal α-galactosidase from fruiting bodies of a wild mushroom Leucopaxillus tricolor.

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 Hg2+ , Fe3 , Cu2+ , Cd2+ , and Mn2+ 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.

Purification and Characterization of a Novel Protease from the Inky Cap Mushroom, Coprinopsis atramentaria (Agaricomycetes).

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.

Profiling of Intra- and Extracellular Enzymes Involved in Fructification of the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

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.

Purification and characterization of a novel metalloprotease from fruiting bodies of Oudemansiella radicata.

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 Km 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 Cd2+, Hg2+, Cu2+, Pb2+ and Fe3+ ions, but was enhanced by K+, Mn2+ and Fe2+ ions. The marked suppression of the protease activity by EDTA indicates that the protease is a metalloprotease.

Purification and partial characterization of a fibrinolytic enzyme from the fruiting body of the medicinal and edible mushroom Pleurotus ferulae.

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.