Purification a laccase exhibiting dye decolorizing ability from an edible mushroom Russula virescens

A novel laccase was purified and characterized from an edible mushroom Russula virescens by using a protocol that comprised ammonium sulfate saturation, ion-exchange chromatography on diethylaminoethyl-cellulose, carboxymethyl-cellulose and quaternary amine-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was a monomeric protein with a molecular mass of 69 kDa. Its N-terminal amino acid sequence was AIGPTAELVV which demonstrated partial sequence homology to those of previously published laccases. Six peptide sequences of the purified laccase were determined by liquid chromatography and linear ion trap quadrupole mass spectrometry. Its optimum pH and temperature were 2.2 and 60 °C, respectively. The laccase was inhibited by inhibitors and several metal ions including Cu²⁺ ions. The laccase degraded various phenolic compounds and the Km toward both 2,7-azinobis (3-ethylbenzothia-zolone-6-sulfonic acid) diammonium salt and dimethylphthalate was 0.1 mM. Moreover, the purified laccase decolorizes a large variety of dyes comprising laboratory dyes such as Bromothymol Blue, Eriochrome black T and Malachite Green and textile dyes such as Reactive Brilliant Blue and Reactive Blue R.     

Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes,and decolorization of chemically different dyes

A laccase (EC 1.10.3.2) was isolated from the culture filtrate of Lentinula edodes. The enzyme was purified to a homogeneous preparation using hydrophobic, anion-exchange, and size-exclusion chromatographies. SDS-PAGE analysis showed the purified laccase, Lcc 1, to be a monomeric protein of 72.2 kDa. The enzyme had an isoelectric point of around pH 3.0. The optimum pH for enzyme activity was around 4.0, and it was most active at 40 degrees C and stable up to 35 degrees C. The enzyme contained 23.8% carbohydrate and some copper atoms. The enzyme oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, p-phenylendiamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol, and ferulic acid, but not veratryl alcohol, tyrosine, and beta-(3,4-dihydroxyphenyl) alanine. The N-terminal amino acid sequence of Lcc 1 showed close homology to the N-terminal sequences determined for laccases from Phlebia radiata, Trametes villosa, and Trametes versicolor, but only low similarity was observed to a previously reported laccase from L. edodes. Lcc 1 was effective in the decolorization of chemically different dyes - Remazole Brilliant Blue R, Bromophenol Blue, methyl red, and Naphtol Blue Black - without any mediators, but the decolorization of two dyes - red poly(vinylamine)sulfonate-anthrapyridone dye and Reactive Orange 16 - did require some redox mediators.     

Isolation of a new ribonuclease from fruiting bodies of the silver plate mushroom Clitocybe maxima

A ribonuclease, with an N-terminal sequence exhibiting some homology to ribonuclease from Pleurotus ostreatus (Family Pleurotaceae), has been purified from fruiting bodies of the silver plate mushroom Clitocybe maxima (Family Tricholomataceae). However, there is little resemblance between the N-terminal sequences of ribonucleases from various Pleurotus species, and a lesser extent of resemblance between ribonucleases from C. maxima and Pleurotus tuber-regium. No structural relationship exists between ribonuclease from C. maxima, and those from Volvariella volvacea, Lentinus edodes and Irpex lacteus. The purification protocol involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose, and fast protein liquid chromatography on Superdex 75. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. It exhibited a molecular mass of 17.5 kDa in both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It manifested roughly the same ribonucleolytic potency toward poly A and poly G followed by poly U. Its activity toward poly C was, by comparison, meager. The temperature and pH required for its optimal activity were, respectively, 70 degrees C and 6.5-7.0.     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from the edible mushroom Schizophyllum commune

An N-acetyl-D-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-D-galactosamine. It was stable at 55 degrees C for 30 min and at pH 3-10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 angstroms resolution.     

Purification ofAgaricus bisporus extracellular laccase from mushroom compost

Summary The extra-cellular laccase of the edible mushroomAgaricus bisporus was purified from non-axenic cultures of the fungus on mushroom compost. Quantities of up to 100 mg of pure enzyme were obtained from single purification runs. Purity of the enzyme protein was comparable to that previously obtained from axenic liquid culture supernatants     

Purification and characterization of phytase with a wide pH adaptation from common edible mushroom Volvariella volvacea (Straw mushroom)

A novel phytase with a molecular mass of 14 kDa was isolated from fresh fruiting bodies of the common edible mushroom Volvariella volvacea (Straw mushroom). The isolation procedure involved successive chromatography on DEAE-cellulose, CM-cellulose, Affi-gel blue gel, Q-Sepharose and Superdex-75. The enzyme was a monomeric protein and was unadsorbed on DEAE-cellulose, CM-cellulose and Affi-gel blue gel, but was adsorbed on Q-Sepharose. The enzyme was purified 51.6-fold from the crude extract with 25.9% yield. Its N-terminal amino acid sequence GEDNEHDTQA exhibited low homology to the other reported phytases. The optimal pH and temperature of the purified enzyme was 5 and 45 degrees C, respectively. The enzyme was quite stable over the pH range of 3.0 to 9.0 with less than 30% change in its activity, suggesting that it can be used in a very wide pH range. The enzyme exhibited broad substrate selectivity towards various phosphorylated compounds, but lacked antifungal activity against tested plant pathogens.     

Purification of a novel extracellular laccase from solid-state culture of the edible mushroom <Emphasis Type="Italic">Lentinula edodes</Emphasis>

The laccases (EC 1.10.3.2) secreted into solid-state culture by Lentinula edodes were analyzed. The fungus secreted at least two laccases in the solid-state culture. One laccase was purified to a homogeneous preparation using anion-exchange, hydrophobic, and size-exclusion chromatography. SDS-PAGE analysis showed that the purified laccase, Lcc6, was a monomeric protein of 58.5 kDa. The optimum pH for enzyme activity was about 3.5, and the laccase was most active at 40°C. The N-terminal amino acid sequence of Lcc6 did not correspond to the sequence of Lcc1, which was previously purified from L. edodes. Lcc6 had decolorization activity to some chemical dyes.     

Extracellular laccase produced by an edible basidiomycetous mushroom, Grifola frondosa: purification and characterization

A major laccase isozyme (Lac 1) was isolated from the culture fluid of an edible basidiomycetous mushroom, Grifola frondosa. Lac 1 was revealed to be a monomeric protein with a molecular mass of 71 kDa. The N-terminal amino acid sequence of Lac 1 was highly similar to those of laccases of some other white-rot basidiomycetes. Lac 1 showed the typical absorption spectrum of a copper-containing enzyme. The enzyme was stable in a wide pH range (4.0 to 10.0), and lost no activity up to 60 °C for 60 min. The optimal pH of the enzyme activity varied among substrates. The K(m) values of Lac 1 toward 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol, catechol, and 3,4-dihydroxy-L-phenylalanine were 0.0137 mM, 0.608 mM, 0.531 mM, 2.51 mM, and 0.149 mM respectively. Lac 1 activity was remarkably inhibited by the chloride ion, in a reversible manner. Lac 1 activity was also inhibited by thiol compounds.     

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.     

Purification and comparative characterization of monophenolase and diphenolase activities from a wild edible mushroom (Macrolepiota gracilenta)

Polyphenol oxidases (PPO) are very important enzymes group in many industrial applications, especially in food, medicine and cosmetics. PPO from Macrolepiota gracilenta, a wild edible mushroom, was purified using a Sepharose 4B-l-tyrosine-p-amino benzoic acid affinity column and characterized in terms of mono- and diphenolase activity. The highest activities for pure enzyme were observed in the presence of PHPPA and DHPPA for monophenolase and diphenolase, respectively. The enzyme showed pH optimum values at 7.0 and 5.0, respectively, for monophenolase and diphenolase activities. K_m values calculated as 0.8 mM for monophenolase and 1 mM for diphenolase activity at the presence of PHPPA and DHPPA as substrate, respectively. V_max values were calculated as 2000 U/mg protein for both activity. Monophenolase and diphenolase activities were conserved approximately 40% and 60%, respectively, in their optimum pH at 4 °C after 5 day incubation. The activities were inhibited most effectively by thiourea. The data obtained from this study showed that this enzyme could be useful for some industrial purposes.     

Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes

Laccase is a blue copper oxidase with multiple copper ions and widely distributed in higher plant and fungi. To date, numerous fungal laccases have been reported by many researchers. In present work, a new laccase gene, named CcLCC5I, from Coprinus cinereus was synthesized chemically according to the yeast bias codon and integrated into Pichia pastoris GS115 genome by electroporation. SDS-PAGE analysis showed that the recombinant laccase has a molecular mass of approximately 56.8 kDa. Its biochemical properties was carried out using substrate 2-2^′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was showed that the optimum pH and temperature of the laccase is 3.0 and 55 °C, respectively. Except for copper ions, most metal ions inhibited the laccase activity at a high concentration about 10 mM. Sodium sulfite can also highly inhibit laccase activity whereas EDTA had no inhibitory effect on the laccase activity. The CcLCC5I have high ability to decolor not only azo but also aryl methane dyes. The recombinant laccase decolored 44.6 % orange G, 54.8 % Crystal Violet, and 87.2 % Malachite green at about 2.6 h. The novel laccase may be a good candidate for breeding engineering strains used in the treatment of industrial effluent containing azo and aryl methane dyes.     

Isolation and characterization of a novel angiotensin I-converting enzyme inhibitory peptide derived from the edible mushroom Tricholoma giganteum

The fruiting body of Tricholoma giganteum has many pharmaceutical uses and has long been utilized as a home remedy in Asia. This study describes the extraction and characterization of the first angiotensin I-converting enzyme (ACE) inhibitory peptide from T. giganteum. The maximum ACE inhibitory activity (IC50: 0.31 mg) was obtained when the fruiting body of T. giganteum was extracted with distilled water at 30 degrees C for 3 h. After the purification of ACE inhibitory peptides with ultrafiltration, Sephadex G-25 column chromatography, and reverse-phase HPLC, an active fraction with an IC50 of 0.04 mg and a yield of 0.3% was obtained. The ACE inhibitory peptide was a novel tripeptide, showing very low similarity to other ACE inhibitory peptide sequences, and was sequenced as Gly-Glu-Pro. The purified ACE inhibitor from T. giganteum competitively inhibited ACE, and it maintained inhibitory activity even after incubation with proteases. ACE inhibitor from T. giganteum showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR), at a dosage of 1 mg/kg.     

Isolation and characterization of a novel mycovirus infecting an edible mushroom,Grifola frondosa

Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.     

Heterologous expression of two minor laccase isozyme cDNAs from the edible mushroom Grifola frondosa

Two cDNAs encoding the minor laccase isozymes (Lac2 and Lac3) of Grifola frondosa were cloned, characterized, and expressed in Pichia pastoris. The recombinant Lac2 (rLac2) was stable at pH 6.0, whereas the recombinant Lac3 (rLac3) was stable in a broad pH range (pH 4.0–8.0). In addition, rLac2 and rLac3 showed the highest catalytic efficiency (k_cat/K_m) for 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid).     

Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces

A novel laccase from the ascomycete Melanocarpus albomyces was purified and characterised. The enzyme was purified using anion exchange chromatography, hydrophobic interaction chromatography and gel filtration, and the purified laccase was biochemically characterised. It had activity towards typical substrates of laccases including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), dimethoxyphenol, guaiacol, and syringaldazine. The laccase showed good thermostability and it had a pH optimum at neutral pH, both unusual properties for most known fungal laccases. The activity of the laccase from M. albomyces was highest at 60-70 degrees C. With guaiacol and syringaldazine the pH optima were rather broad: 5-7.5 and 6-7, respectively. It retained 50% of its activity after 5 h incubation at 60 degrees C. The molecular weight of the laccase was about 80 kDa and the isoelectric point 4.0. The ultraviolet-visible absorption and electron paramagnetic resonance spectra of the purified laccase indicated that the typical three types of copper were present.