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.

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.

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.

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.

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.

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.

Characterization of a novel laccase purified from the fungus Hohenbuehelia serotina and its decolourisation of dyes.

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 ß-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.

Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

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.

ISOLATION AND PROPERTIES OF POLYPHENOL OXIDASE FROM BASIDIOCARPS OF Lactarius pergamenus Fr. (Fr.) FUNGI.

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 ß-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.

Lentinan degradation in the Lentinula edodes fruiting body during postharvest preservation is reduced by downregulation of the exo-ß-1,3-glucanase EXG2.

Lentinan from Lentinula edodes fruiting bodies (shiitake mushrooms) is a valuable ß-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-ß-1,3-glucanase, exg2, in L. edodes using RNA interference. In the wild-type strain, ß-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.

Selenium enrichment on Cordyceps militaris link and analysis on its main active components.

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.

Purification and characterization of a novel RNase with antiproliferative activity from the mushroom Lactarius flavidulus.

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.

Partial purification and characterization of polyphenoloxidase from culinary-medicinal Royal Sun mushroom (the Himematsutake), Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae).

The Royal Sun mushroom, the Himematsutake culinary-medicinal mushroom, Agaricus brasiliensis has several polyphenoloxidase activities in a broad sense. Here we report the partial purification of tyrosinase-type polyphenoloxidase (PPO). PPO is purified from A. brasiliensis without browning using a two-phase partitioning with Triton X-114 and ammonium sulfate fractionation. Partially denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) staining with L-3,4-dihydroxyphenylalanine was performed and the indicated molecular sizes were approximately 70 kDa and 45 kDa. The purified enzyme is in its latent state and can be activated maximally in the presence of 1.6 mM sodium dodecyl sulfate (SDS). This enzyme catalyzes two distinct reactions, monophenolase and diphenolase activity, and the monophenolase activity showed a lag time typical of polyphenoloxidase. The K(m) value for 4-tert-butylcatechol was quite similar in the presence and absence of SDS, but the apparent V(max) value was increased 2.0-fold by SDS. Mimosine was a typical competitive inhibitor with K(i) values of 138.2 microM and 281.0 microM n the presence and absence of SDS, respectively.

A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.

A novel ribonuclease with potent HIV-1 reverse transcriptase inhibitory activity from cultured mushroom Schizophyllum commune.

A 20-kDa ribonuclease (RNase) was purified from fresh fruiting bodies of cultured Schizophyllum commune mushrooms. The RNase was not adsorbed on Affi-gel blue gel but adsorbed on DEAE-cellulose and CM-cellulose. It exhibited maximal RNase activity at pH 6.0 and 70°C. It demonstrated the highest ribonucleolytic activity toward poly (U) (379.5 µ/mg), the second highest activity toward poly (C) (244.7 µ/mg), less activity toward poly (A) (167.4 µ/mg), and much weaker activity toward poly (G) (114.5 µ/mg). The RNase inhibited HIV-1 reverse transcriptase with an IC(50) of 65 µM. No effect on [(3)H-methyl]-thymidine uptake by lymphoma MBL2 cells and leukemia L1210 cells was observed at 100 µM concentration of the RNase. A comparison of RNases from S. commune and Volvariella volvacea revealed that they demonstrated some similarities in N-terminal amino acid sequence, optimum pH and polyhomoribonucleotide specificity. However, some differences in chromatographic behavior and molecular mass were observed.

A novel metalloprotease from the wild basidiomycete mushroom Lepista nuda.

A 20.9-kDa metalloprotease was isolated from dried fruiting bodies of the wild basidiomycete mushroom Lepista nuda. The N-terminal amino acid sequence of the protease was seen to be ATFVLTAATNTLFTA, thus displaying no similarity with the sequences of previously reported metalloproteases. The protease was purified using a procedure that entailed ion-exchange chromatography on CM-Cellulose, Q-Sepharose, and Mono S, and FPLC-gel filtration on Superdex 75. The protease functioned at an optimum pH of 7.0 and an optimum temperature of 50 degrees C. It was also noted that the protease demonstrated a proteolytic activity of 1,756 U/mg toward casein. The Km of the purified protease toward casein was 6.36 mg/ml at a pH of 7.0 and with a temperature of 37 degrees C, whereas the Vmax was 9.11 microgram ml(-1) min(-1). The activity of the protease was adversely affected by EDTA-2Na, suggesting that it is a metalloprotease. PMSF, EGTA, aprotinin, and leupeptin exerted no striking inhibitory effect. The activity of the protease was enhanced by Fe2+, but was curtailed by Cd2+, Cu2+, Hg2+, Pb2+, Zn2+, and Fe3+ ions. The protease also exhibited inhibitory activity against HIV-1 reverse transcriptase with an IC50 value of 4.00 micrometer. The IC50 values toward hepatoma Hep G2 and leukemia L1210 cells in vitro were 4.99 micrometer and 3.67 micrometer, respectively.

A laccase with antiproliferative activity against tumor cells from an edible mushroom, white common Agrocybe cylindracea.

A laccase, with HIV-1 reverse transcriptase inhibitory activity (IC(50)=12.7 µM) and antiproliferative activity against HepG2 cells (IC(50)=5.6 µM) and MCF7 cells (IC(50)=6.5 µM), was purified from fresh fruiting bodies of the edible white common Agrocybe cylindracea mushroom. The laccase, which had a novel N-terminal sequence, displayed a molecular mass of 58 kDa within the range reported for most other mushroom laccases. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, SP-Sepharose, and Q-Sepharose and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but unadsorbed on SP-Sepharose. Its optimum pH was pH 3-4 and its optimum temperature was 50°C. The activity of the isolated laccase differed from one substrate to another. The ranking was ABTS>N,N-dimethyl-1,4-phenylenediamine>hydroquinone>catechol>2-methylcatechol>pyrogallol.

An antiproliferative ribonuclease from fruiting bodies of the wild mushroom Russula delica.

An antiproliferative ribonuclease with a new N-terminal sequence was purified from fruiting bodies of the edible wild mushroom Russula delica in this study. This novel ribonuclease was unadsorbed on DEAE-cellulose, but absorbed on SP-Sepharose and Q-Sepharose. It had a molecular mass of 14 kDa as judged by fast protein liquid chromatography on Superdex 75 and SDS-polyacrylamide gel electrophoresis. Its optimal pH and optimal temperature were pH 5 and 60 degrees , respectively. The ranking of its activity toward various polyhomoribonucleotides was poly C > poly G > poly A > poly U. It could inhibit proliferation of HepG2 and MCF-7 cancer cells with an IC50 value of 8.6 microM and 7.2 microM, respectively. It was devoid of antifungal and HIV-1 reverse transcriptase inhibitory activity.

A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji.

A 14.5-kDa ribonuclease, with an optimum pH of 6 and a temperature optimum at 70 degrees C, was isolated from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. It was purified by ion exchange chromatography on DEAE cellulose, Q Sepharose, and SP Sepharose, followed by FPLC gel filtration on Superdex 75, and was adsorbed on all three ion exchangers. It showed the highest ribonucleolytic potency toward poly (U), 25% as much activity toward poly (C), and undetectable activity toward poly (A) and poly (G). Its ribonucleolytic activity at 100 degrees C was similar to that at 20 degrees C. It suppressed proliferation of hepatoma HepG2 cells and breast cancer MCF7 cells with an IC(50) of 10 and 6.2 microM, respectively. It inhibited the activity of HIV-1 reverse transcriptase with an IC(50) of 7.2 microM.

[Effect of exogenous zinc addition on cell protective enzyme activities in the fruit bodies of Lentinus giganteus].

OBJECTIVE: The effects of exogenous Zn addition on cell protective enzyme activities in the fruit bodies of Lentinus giganteus were studied. METHODS: ZnSO4 was used as exogenous Zn and added into culture medium. The final Zn concentrations in culture media were 0, 10, 20, 30, 40, 50 mg/kg respectively. The activities of superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), malondialchehyche (MDA) content and soluble protein content in the fruit bodies were analyzed by spectrophotometry; catalase (CAT) was determined by potassium permanganate titration. RESULTS: The content of soluble protein and SOD, POD and CAT activities in the fruit bodies of L. giganteus were significantly increased (P < 0.01), but PPO activity (P < 0.01) and MDA content (P < 0.05) was significantly decreased in the treatment of 30 mg/kg Zn concentration. The content of soluble protein and SOD, POD and CAT activities showed a decreasing trend with the increase of Zn concentration, but MDA content was significantly increased (P < 0.01 and P < 0.05). CONCLUSION: High Zn concentration caused the increase of MDA contents and the decrease of SOD, POD and CAT activities in the fruit body of L. giganteus. It will destroy the protective enzyme system, cause the accumulation of free radicals and thus intensify membrane lipid peroxidation. Appropriate Zn concentration improved the protective enzyme activities, and lightened the harm of membrane from lipid peroxidation.