RESUMO
The present work describes the purification of an enzyme capable of degrading punicalagin. The enzyme was produced by Aspergillus niger GH1 by solid-state fermentation, and the enzyme production was induced by using ellagitannins as the sole carbon source. The purification steps included the concentration by lyophilization, desalting, anionic exchange, and gel filtration chromatography. The enzyme kinetic constants were calculated by using punicalagin, methyl gallate, and sugar beet arabinans. The molecular mass of the protein was estimated by SDS-PAGE. The identified bands were excised and digested using trypsin, and the peptides were submitted to HPLC-MS/MS analysis. The docking analysis was conducted, and a 3D model was created. The purification fold increases 75 times compared with the cell-free extract. The obtained Km values were 0.053 mM, 0.53% and 6.66 mM for punicalagin, sugar beet arabinans and methyl gallate, respectively. The optimal pH and temperature for the reaction were 5 and 40 °C, respectively. The SDS-PAGE and native PAGE analysis revealed the presence of two bands identified as α-l-arabinofuranosidase. Both enzymes were capable of degrading punicalagin and releasing ellagic acid.
RESUMO
The role and mechanism of elagitannase is misunderstood because it exhibited different activities due to the low purity or complexity of substrates, and there is no available information about the biochemical, physicochemical and molecular characteristics of the enzyme. This study was aimed to obtain enzymatic extracts by Aspergillus niger GH1 in solid-state fermentation, using dextrose and ellagitannins as inducers of ellagitannase. Protein and bioinformatic analysis were performed to identify the protein sequence expressed in terms of culture conditions. The presence of ellagitannins increased ellagitannase activity 1143-fold compared to dextrose. The higher ellagitannase activity was found at 18 h of culture (1143.30 U g-1PE). Three groups of proteins were identified in both cultures: ß-glucosidase, phospholipase C, and triacylglycerol lipase. However, only phospholipase C was overexpressed with ellagitannins as inducers, showing the most spontaneous reaction with punicalagin (ΔG -8.56). These results suggest that phospholipase could be involved in ellagitannins biosynthesis.
Assuntos
Ácido Elágico , Taninos Hidrolisáveis , Aspergillus niger/metabolismo , Fermentação , Taninos Hidrolisáveis/metabolismoRESUMO
The genus Aspergillus is ubiquitous in nature and includes various species extensively exploited industrially due to their ability to produce and secrete a variety of enzymes and metabolites. Most processes are performed in submerged fermentation (SmF); however, solid-state fermentation (SSF) offers several advantages, including lower catabolite repression and substrate inhibition and higher productivity and stability of the enzymes produced. This study aimed to explain the improved metabolic behavior of A. brasiliensis ATCC9642 in SSF at high glucose concentrations through a proteomic approach. Online respirometric analysis provided reproducible samples for secretomic studies when the maximum CO2 production rate occurred, ensuring consistent physiological states. Extracellular extracts from SSF cultures were treated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS. Of 531 sequences identified, 207 proteins were analyzed. Twenty-five were identified as the most abundant unregulated proteins; 87 were found to be up-regulated and 95 were down-regulated with increasing glucose concentration. Of the regulated proteins, 120 were enzymes, most involved in the metabolism of carbohydrates (51), amino acids (23), and nucleotides (9). This study shows the high protein secretory activity of A. brasiliensis under SSF conditions. High glucose concentration favors catabolic activities, while some stress-related proteins and those involved in proteolysis are down-regulated.
Assuntos
Aspergillus/metabolismo , Fermentação , Glucose/metabolismo , Aspergillus/enzimologia , Metabolismo dos Carboidratos , Dióxido de Carbono/metabolismo , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Espectrometria de Massas , Metabolismo/efeitos dos fármacos , Proteômica/métodosRESUMO
Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response.
Assuntos
Antígenos de Fungos/imunologia , Parede Celular/imunologia , Glicoproteínas/imunologia , Imunidade Celular/efeitos dos fármacos , Sporothrix/imunologia , Esporotricose/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Fungos/administração & dosagem , Antígenos de Fungos/química , Parede Celular/química , Citocinas/genética , Citocinas/imunologia , Expressão Gênica , Glicoproteínas/administração & dosagem , Glicoproteínas/química , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Especificidade da Espécie , Esporos Fúngicos/química , Esporos Fúngicos/imunologia , Esporos Fúngicos/patogenicidade , Sporothrix/química , Sporothrix/patogenicidade , Esporotricose/genética , Esporotricose/microbiologia , Células Th1/imunologia , Células Th1/microbiologia , Equilíbrio Th1-Th2 , Células Th17/imunologia , Células Th17/microbiologiaRESUMO
The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.
Assuntos
Antígenos de Fungos/análise , Parede Celular/imunologia , Sporothrix/imunologia , Antígenos de Fungos/química , Antígenos de Fungos/imunologia , Parede Celular/química , Eletroforese em Gel Bidimensional , Humanos , Immunoblotting , Peso Molecular , Sporothrix/químicaRESUMO
Significant differences on structure, stability, and catalytic properties of tannase were found when this enzyme was produced under solid-state and submerged fermentations (SSF and SmF) by Aspergillus niger. The specific activity was 5.5 times higher on SSF than in SmF. Significant differences in isoelectric points of tannases were found. The pH optima for both types of enzyme was found at 6 and the pH stability of SSF and SmF tannase were at 6 and 5-8, respectively. The optimal temperature range was from 50 to 60 °C for SmF tannase and 60 °C for SSF tannase, and both enzyme types showed tolerance to high temperatures (60-70 °C). The SSF tannase showed a major specificity for methyl gallate substrate while SmF tannase for tannic acid. All metal ions tested, had an activity inhibition from 30-46% on SSF tannase. SDS-PAGE analysis as well as gel localization studies of both SSF and SmF purified tannases showed a single band with a molecular weight of 102 and 105 kDa, respectively. Different levels of glycosylation were found among SSF and SmF purified tannases. This is the first report about structural differences among tannase produced under SSF and SmF and this study provides basis for explanation of the stability and catalytic differences observed previously for this two tannase types.
Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Eletroforese em Gel de Poliacrilamida , Fermentação/fisiologia , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
In the last years, tannase has been the subject of a lot of studies due to its commercial importance and complexity as catalytic molecule. Tannases are capable of hydrolyzing complex tannins, which represent the main chemical group of natural anti-microbials occurring in the plants. The general outline of this work includes information of the substrates, the enzyme, and the applications. This review considers in its introduction the concepts and history of tannase and explores scientific and technological aspects. The "advances" trace the route from the general, molecular, catalytic, and functional information obtained under close to optimal conditions for microbial production through purification, description of the enzyme properties, and the commercial applications to the "perspectives" including expression studies, regulation, and potential uses; aspects related to the progress in our understanding of tannin biodegradation are also included.