RESUMO
The characterization of a novel protease from Amanita virgineoides is described. The A. virgineoides protease was purified to homogeneity using Q-Sepharose, carboxymethyl-cellulose, diethylaminoethyl-cellulose, and a gel filtration step on Superdex 75. The molecular mass of the purified protease was estimated to be 16.6 kDa. The protease was purified 32.1-fold, and its specific activity was 301.4 U/mg. The optimum pH was 4.0, and the optimum temperature was 50 °C. Kinetic constants (Km , Vmax ) were determined under the optimum reaction conditions, with Km and Vmax , being 3.74 mg/mL and 9.98 µg mL-1 Min-1 , respectively. The activity of the protease was curtailed by Cu2+ , Pb2+ , Fe3+ , Cd2+ , and Hg2+ ions but enhanced by Mg2+ , Ca2+ , and K+ ions at low concentrations. The protease activity was adversely affected by ethylene diamine tetraacetic acid, suggesting that it is a metalloprotease. Four peptide sequences were obtained from liquid chromatography-tandem mass spectrometry, including KQALSGIR, TIAMDGTEGLVR, VALTGLTVAEYFR, and AGAGSATLSMAYAGAR, which showed 86%, 64%, 60%, and 75% identity with peptides of Hypsizygus marmoreus, Dacryopinax sp. DJM-731 SS1, Trametes versicolor FP-101664 SS1, and Paxillus involutus ATCC 200175, respectively. The newly isolated protease showed good hydrolytic activity and biochemical characteristics, which expanded the knowledge of biologically active proteins and provided further insight on this poisonous fungus.
Assuntos
Amanita/enzimologia , Proteínas Fúngicas/metabolismo , Metaloproteases/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Hidrólise , Metaloproteases/química , Metaloproteases/isolamento & purificação , Peso MolecularRESUMO
The characterization of three laccase isoforms from Pleurotus nebrodensis is described. Isoenzymes Lac1, Lac2 and Lac3 were purified to homogeneity using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a gel filtration step on Superdex 75. The molecular weights of the purified laccases were estimated to be 68, 64 and 51 kDa, respectively. The isoenzymes demonstrated the same optimum pH at 3.0 but slightly different temperature optima: 50-60 °C for Lac1 and Lac3 and 60 °C for Lac2. Lac2 was always more stable than the other two isoforms and exposure to 50 °C for 120 min caused 30% loss in activity. Lac2 was relatively less stable than the other two isoforms when exposed to the pH range of 3.0-8.0 for 24 h, but inactivation only occurred initially, with around 70% residual activity being maintained during the whole process. Oxidative ability towards aromatic compounds varied substantially among the isoforms and each of them displayed preference toward some substrates. Kinetic constants (Km, Kcat) were determined by using a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay, with Lac3 showing the best affinity and Lac2 displaying the highest catalytic efficiency. Amino acid sequences from peptides derived from digestion of isoenzymes showed great consistency with laccases in the databases.
Assuntos
Lacase/isolamento & purificação , Lacase/metabolismo , Pleurotus/enzimologia , Sequência de Aminoácidos , Ativação Enzimática , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Isoenzimas/isolamento & purificação , Lacase/química , Peso Molecular , Pleurotus/químicaRESUMO
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.
Assuntos
Basidiomycota/enzimologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Ribonucleases/isolamento & purificação , Ribonucleases/farmacologia , Sequência de Aminoácidos , Cromatografia em Gel , Cromatografia por Troca Iônica , DEAE-Celulose , Eletroforese em Gel de Poliacrilamida , Carpóforos/enzimologia , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Ribonucleases/metabolismo , TaiwanRESUMO
An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS), indicating the importance of tryptophan residue(s) at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.
Assuntos
Proteínas Fúngicas , Oligossacarídeos/química , Rafinose/química , Tricholoma/enzimologia , alfa-Galactosidase , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Especificidade por Substrato , alfa-Galactosidase/química , alfa-Galactosidase/isolamento & purificaçãoRESUMO
An 86-kDa homodimeric angiotensin I-converting enzyme (ACE) inhibitory protein designated as LTP was isolated from fruit bodies of the mushroom Leucopaxillus tricolor. The isolation procedure involved ultrafiltration through a membrane with a molecular weight cutoff of 10-kDa, ion exchange chromatography on Q-Sepharose, and finally fast protein liquid chromatography-gel filtration on Superdex 75. LTP exhibited an IC50 value of 1.64 mgâmL-1 for its ACE inhibitory activity. The unique N-terminal amino acid sequence of LTP was disclosed by Edman degradation to be DGPTMHRQAVADFKQ. In addition, seven internal sequences of LTP were elucidated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results of the Lineweaver-Burk plot suggested that LTP competitively inhibited ACE. Both LTP and the water extract of L. tricolor exhibited a clear antihypertensive effect on spontaneously hypertensive rats.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Anti-Hipertensivos/isolamento & purificação , Carpóforos/química , Proteínas Fúngicas/isolamento & purificação , Hipertensão/tratamento farmacológico , Peptidil Dipeptidase A/metabolismo , Agaricales/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Ligação Competitiva , Pressão Sanguínea/efeitos dos fármacos , Cromatografia em Gel , Cromatografia por Troca Iônica , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Ratos , Ratos Endogâmicos SHRRESUMO
To date, only a few steroids have been isolated from the mushroom Stropharia rugosoannulata which can be cultivated. In this paper, a novel lectin (SRL) with a molecular weight of 38 kDa, and a unique IKSGVYRIVSWQGALGPEAR N-terminal sequence was isolated from S. rugosoannulata, which represents the first protein isolated from the mushroom. The purification methods included (NH4)2SO4 precipitation, ion exchange chromatography on CM-cellulose, Q-Sepharose, and SP-Sepharose, and gel- filtration on Superdex-75. The lectin was adsorbed on all three types of ion exchangers and was purified more than 450-fold. The lectin was stable below 70 °C (with half of the activity preserved at 80 °C), and in the presence of NaOH and HCl solutions up to a concentration of 12.5 mM and 25 mM, respectively. The hemagglutinating activity of SRL was inhibited by inulin. Cd2+ and Hg2+ ions strongly reduced the hemagglutinating activity at concentrations from 1.25 mM to 10 mM. SRL exhibited anti-proliferative activity toward both hepatoma Hep G2 cells and leukemia L1210 cells, with an IC50 of 7 µM and 19 µM, respectively. The activity of HIV-1 reverse transcriptase could also be inhibited by SRL, with an IC50 of 10 µM.
Assuntos
Agaricales/química , Lectinas/isolamento & purificação , Lectinas/farmacologia , Sequência de Aminoácidos , Animais , Carboidratos/farmacologia , Cátions/farmacologia , Eletroforese em Gel de Poliacrilamida , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , Hemaglutinação/efeitos dos fármacos , Células Hep G2 , Humanos , Ácido Clorídrico/farmacologia , Concentração Inibidora 50 , Lectinas/química , Camundongos , Dados de Sequência Molecular , Coelhos , Hidróxido de Sódio/farmacologia , TemperaturaRESUMO
A 36-kDa protein, with an N-terminal sequence highly homologous to polygalacturonase (PG) inhibiting proteins, was isolated from small brown-eyed cowpea seeds. The protein was unadsorbed on diethylaminoethyl cellulose but adsorbed on both Affi-gel blue gel and SP-sepharose. It inhibited mycelial growth in the fungus Mycosphaerella arachidicola with an half-maximal (50%) inhibitory concentration (IC50 ) of 3.3 µM. It reduced [methyl-(3) H] thymidine incorporation into MBL2 lymphoma and L1210 leukemia cells with an IC50 of 7.4 and 5.4 µM, respectively. It inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with an IC50 of 12.9 µM. However, it did not inhibit PG. The potent antifungal and antitumor activities of the protein suggest that it can be developed into an antifungal agent for combating M. arachidicola invasion in crops and an agent for cancer therapy in humans.
Assuntos
Antifúngicos/isolamento & purificação , Fabaceae/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Inibidores da Transcriptase Reversa/isolamento & purificação , Sementes/química , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fungos/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Proteínas de Plantas/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
A novel polysaccharide, Heimioporus retisporus Polysaccharide (HRP) was extracted from the edible mushroom Heimioporus retisporus. HRP had weight-average molecular weight 1,949 kDa and number-average molecular weight 873 kDa, and its major components were arabinose (0.71%), galactose (12.93%), glucose (49.00%), xylose (8.59%), mannose (17.78%), and glucuronic acid (10.99%). Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy revealed that HRP was composed of 1,3-linked ß-D-glucose, 1,6-linked ß-D-mannose, 1,6-linked ß-D-galactose, 1,4-linked ß-D-galactose, 1,4-linked ß-D-xylose, and 1,5-linked α-L-arabinose. Thermogravimetric analysis indicated that degradation temperature (T0) of HRP was 200°C. In an STZ-induced diabetic mouse model, oral administration of HRP (40 mg/kg/d) for 28 days significantly reduced blood glucose levels, and reduced heart organ index by decreasing expression of IL-6 and TNF-α. Our findings indicate hypoglycemic effect of HRP, and its potential application as a hypoglycemic agent.
RESUMO
An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 µM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.
Assuntos
Cannabis/enzimologia , Rafinose/isolamento & purificação , Sementes/enzimologia , alfa-Galactosidase/química , Bromosuccinimida/química , Cromatografia Líquida/métodos , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Metais Pesados/farmacologia , Peso Molecular , Nitrofenilgalactosídeos/química , Rafinose/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triptofano/análise , alfa-Galactosidase/antagonistas & inibidores , alfa-Galactosidase/isolamento & purificaçãoRESUMO
An acidic α-galactosidase designated as PDGI (Pleurotus djamor α-galactosidase) was purified to homogeneity with 290-fold purification and a specific activity of 52.18 units/mg by means of ion exchange chromatography and gel filtration chromatography. PDGI is a monomeric protein exhibiting a molecular mass of 60kDa in SDS-PAGE and gel filtration. The optimum pH and temperature of the enzyme with pNPGal as substrate were 5.0 and 53.5°C, respectively. It displayed great pH stability within the pH range 3.0-10.0. Besides, the enzyme harbored remarkable resistance to acid protease and varying degrees of tolerance to other proteases: trypsin>collagenase Type-I>α-chymotrypsin neutral protease>proteinaseK. It was strongly inhibited by K+, Cd2+, Cu2+, Hg2+, Al3+, Fe3+ and Ag + ions. The chemical modification reagents diethypyrocarbonate (DEPC), 2,3-butanedione (DIC) and trinitrophenol (TNBS) increased the activity of PDGI 1.5-fold whereas N-bromosuccinimide (NBS) and parachloro-mercuri-benzoate (PCMB) drastically suppressed its activity. PDGI displayed activity toward stachyose and raffinose. The Km values for hydrolysis of pNPGal, stachyose and raffinose were 0.76, 7.63 and 6.29mM, respectively. Furthermore, PDGI degraded raffinose and sthachyose. These results suggest that PDGI has great potential for elimination of the non-digestible and flatulence-causing oligosaccharides stachyose and raffinose from legumes.
Assuntos
Proteínas Fúngicas/química , Pleurotus/enzimologia , Rafinose/química , alfa-Galactosidase/química , Sequência de Aminoácidos , Quimotripsina/química , Estabilidade Enzimática , Proteínas Fúngicas/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Metais/química , Peso Molecular , Oligossacarídeos/química , Especificidade por Substrato , alfa-Galactosidase/antagonistas & inibidoresRESUMO
A 45-kDa monomeric acidic α-galactosidase with a specific activity of 193.12 units/mg was isolated from the fruiting bodies of Agaricus bisporus. Blast search of internal peptide sequences suggested that it is a member of GH family 27 and it is most similar to hypothetical protein AGABI2DRAFT_70106. The enzyme displayed maximal activity at pH 4.0 and 60°C, respectively. The enzyme remained stable within the pH range 2.0-9.0 but its activity was markedly suppressed in the presence of Cu2+, Hg2+, Fe3+ and Ag+ ions. It displayed resistance to α-chymotrypsin and neutral protease. Moreover, it manifested degradative activity toward both oligosaccharides and polysaccharides. The enzyme manifested Km values of 0.30mM, 10.65mM and 19.21mM, toward pNPGal, stachyose and raffinose respectively. These results suggest that Agaricus bisporus α-galactosidase is a promising candidate for elimination of raffinose oligosaccharides (RFOs) in biotechnological applications.
Assuntos
Agaricus/química , Peptídeo Hidrolases/metabolismo , Rafinose/metabolismo , alfa-Galactosidase/metabolismo , Biocatálise , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Especificidade por Substrato , alfa-Galactosidase/químicaRESUMO
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.
Assuntos
Agaricales/enzimologia , Proteínas Fúngicas/metabolismo , Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Cromatografia por Troca Iônica , Carpóforos/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Metaloproteases/química , Metais/química , Metais/farmacologia , Peso Molecular , Espectrometria de Massas em Tandem , TemperaturaRESUMO
Hypertension is a major risk factor for cardiovascular disease. A crude water extract of the fruiting bodies of a highly prized mushroom Tricholoma matsutakei exerted an antihypertensive action on spontaneously hypertensive rats (SHRs) at a dosage of 400 mg/kg. An angiotensin converting enzyme (ACE) inhibitory peptide with an IC50 of 0.40 µM was purified from the extract and designated as TMP. Its amino acid sequence was elucidated to be WALKGYK through LC-MS/MS analysis. The Lineweaver-Burk plot suggested that TMP was a non-competitive inhibitor of ACE. A short-term assay of antihypertensive activity demonstrated that TMP at the dosage of 25 mg/kg could significantly lower the systolic blood pressure (SBP) of SHRs. TMP exhibited remarkable stability over a wide range of temperatures and pH values. It also demonstrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. The aforementioned activities of TMP were corroborated by utilizing the synthetic peptide. Hence T. matsutake can be used as a functional food to help prevent hypertension- associated diseases.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/farmacologia , Antioxidantes/farmacologia , Peptídeos/farmacologia , Tricholoma/metabolismo , Sequência de Aminoácidos , Animais , Compostos de Bifenilo/antagonistas & inibidores , Compostos de Bifenilo/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Estabilidade de Medicamentos , Sequestradores de Radicais Livres/farmacologia , Carpóforos/metabolismo , Concentração de Íons de Hidrogênio , Hipertensão/fisiopatologia , Hipertensão/prevenção & controle , Peptídeos/isolamento & purificação , Picratos/antagonistas & inibidores , Picratos/metabolismo , Ratos Endogâmicos SHR , TemperaturaRESUMO
Three laccase isoenzymes (Lac1, Lac2 and Lac3) have been purified to homogeneity from Pleurotus nebrodensis in our previous study. Lac2 was shown to be the dominant isoform, capable of oxidizing the majority of laccase substrates and manifesting good thermostability and pH stability. Hence, Lac2 was selected to decolourize structurally different dyes and the colour removal efficiencies of Lac2 and the crude extract of P. nebrodensis were compared. By monitoring the λmax of the reaction system during the course of biotransformation, clear hypsochromic shifts were observed for most of the dyes examined, illustrating that at least one peak disappeared as a result of laccase treatment. In general, Lac2 was more efficient within a short time (1 h) and the crude extract, in general, could achieve similar or even higher efficiency when the duration of treatment was extended to 24 h. Malachite green (MG) was chosen to study the detoxifying potential of Lac2, because of the relatively simple structure and high toxicity of the dye towards microorganisms. The toxicity of MG towards both bacteria (Bacillus subtilis, Bacillus licheniformis, Pseudomonas fluorescens and Escherichia coli) and fungi (Fusarium graminearum and Trichoderma harzianum) was dramatically decreased and the potential mechanism was estimated by GC-MS as to remove four methyl groups firstly and the two newly formed amine groups would be degraded or polymerized further. The present study facilitates an understanding of the application of P. nebrodensis laccases and furnishes evidence for the safety of their utilization in the treatment of wastewater emanating from textile industries.
Assuntos
Corantes/química , Poluentes Ambientais/química , Proteínas Fúngicas/química , Lacase/química , Pleurotus/enzimologia , Corantes de Rosanilina/química , Bacillus licheniformis/efeitos dos fármacos , Bacillus licheniformis/crescimento & desenvolvimento , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Biodegradação Ambiental , Corantes/toxicidade , Misturas Complexas/química , Poluentes Ambientais/toxicidade , Estabilidade Enzimática , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas Fúngicas/isolamento & purificação , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/isolamento & purificação , Lacase/isolamento & purificação , Viabilidade Microbiana/efeitos dos fármacos , Oxirredução , Pleurotus/química , Pseudomonas fluorescens/efeitos dos fármacos , Pseudomonas fluorescens/crescimento & desenvolvimento , Corantes de Rosanilina/toxicidade , Trichoderma/efeitos dos fármacos , Trichoderma/crescimento & desenvolvimentoRESUMO
An alpha-galactosidase was purified from Pseudobalsamia microspora (PMG) to 1224.1-fold with a specific activity of 11,274.5 units/mg by ion-exchange chromatography and gel filtration. PMG is a monomeric protein with a molecular mass of 62 kDa as determined by SDS-PAGE and by gel filtration. Chemical modification using N-bromosuccinimide (NBS) resulted in a complete abrogation of the activity of PMG, suggesting that Trp is an amino acid essential to its activity. The activity was strongly inhibited by Hg(2+), Cd(2+), Cu(2+), and Fe(3+) ions. Three inner peptide sequences for PMG were obtained by liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis. When 4-nitrophenyl α-D-glucopyranoside (pNPGal) was used as substrate, the optimum pH and temperature of PMG were 5.0 and 55 °C, respectively. The Michaelis constant (K m) value of the alpha-galactosidase on pNPGal was 0.29 mM, and the maximal velocity (V max) was 0.97 µmol ml(-1) min(-1). Investigation by thin-layer chromatography (TLC) demonstrated its ability to hydrolyze raffinose and stachyose. Hence, it can be exploited in degradation of non-digestible oligosaccharides from food and feed industries.
Assuntos
Microsporídios/enzimologia , Rafinose/metabolismo , alfa-Galactosidase/metabolismo , Sequência de Aminoácidos , Cromatografia por Troca Iônica , Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Hidrólise , Indicadores e Reagentes , Íons , Cinética , Metais/farmacologia , Microsporídios/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Especificidade por Substrato/efeitos dos fármacos , Temperatura , alfa-Galactosidase/química , alfa-Galactosidase/isolamento & purificaçãoRESUMO
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.
Assuntos
Agaricales/química , Antídotos/farmacologia , Proteínas Fúngicas/farmacologia , Nitrito Redutases/farmacologia , Nitrito de Sódio/intoxicação , Agaricales/enzimologia , Animais , Antídotos/isolamento & purificação , Antídotos/metabolismo , Antídotos/farmacocinética , Carcinógenos/antagonistas & inibidores , Carcinógenos/metabolismo , Dieta , Ensaios Enzimáticos , Fermentação/efeitos dos fármacos , Carpóforos/química , Carpóforos/enzimologia , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacocinética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Metemoglobina/antagonistas & inibidores , Metemoglobina/metabolismo , Camundongos , Nitrito Redutases/isolamento & purificação , Nitrito Redutases/metabolismo , Nitrito Redutases/farmacocinética , Nitrosaminas/antagonistas & inibidores , Nitrosaminas/metabolismo , Ratos Sprague-Dawley , Nitrito de Sódio/metabolismo , Temperatura , Verduras/intoxicaçãoRESUMO
The aim of the present study was to investigate the antioxidant and hepatoprotective effects of water-soluble polysaccharides (RVLWP) and alkali-soluble polysaccharides (RVLAP) from Russula vinosa on carbon tetrachloride (CCl4)-induced acute liver damage in mice. For the in vitro antioxidant activities, RVLWP and RVLAP exhibited potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 = 1.55 ± 0.04 and 3.37 ± 0.21 mg/mL, respectively), hydrogen peroxide scavenging activity (IC50 = 6.07 ± 0.24 and 9.23 ± 0.54 mg/mL, respectively), lipid peroxidation inhibitory effect (IC50 = 0.52 ± 0.095 and 0.86 ± 0.043 mg/mL, respectively), and moderate reducing power and Fe(2+) chelating activity (IC50 = 1.86 ± 0.0036 and 0.22 ± 0.0057 mg/mL, respectively). Ascorbic acid was employed as the standard antioxidant in the present study. For the in vivo hepatoprotective activity, administration of RVLWP and RVLAP (200 mg/kg) significantly prevented the elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in acute liver damage induced by CCl4 and suppressed hepatic malondialdehyde (MDA) formation. Mice treated with RVLWP and RVLAP demonstrated a better profile of antioxidants with augmented activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver. The results suggest that RVLWP and RVLAP protect the liver from CCl4-induced hepatic damage via antioxidant mechanisms.
Assuntos
Agaricales/química , Antioxidantes/administração & dosagem , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Extratos Vegetais/administração & dosagem , Polissacarídeos/administração & dosagem , Substâncias Protetoras/administração & dosagem , Alanina Transaminase/sangue , Animais , Antioxidantes/química , Aspartato Aminotransferases/sangue , Tetracloreto de Carbono/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Glutationa Peroxidase/metabolismo , Humanos , Masculino , Malondialdeído/metabolismo , Camundongos , Fitoterapia , Extratos Vegetais/química , Polissacarídeos/química , Substâncias Protetoras/química , Superóxido Dismutase/metabolismoRESUMO
A novel laccase with a molecular mass of 64 kDa and the N-terminal sequence AIGPDDTINF was isolated from fresh fruiting bodies of the mushroom Pleurotus nebrodensis. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration on Superdex 75. The laccase was adsorbed on DEAE-cellulose and Q-Sepharose, but not on CM-cellulose. It demonstrated an optimal temperature of 70°C. The enzyme activity increased steadily over the temperature range 20°C-70°C. There was only a slight reduction in activity at 80°C. However, all activity disappeared following exposure to 100°C for 10 minutes. The enzyme activity changed only slightly over the pH range 3-5, with the optimum at pH 5, but underwent a precipitous decline when the pH was elevated to 6, and was undetectable at pH 8 and pH 9.