RESUMO
BACKGROUND: Eqolisins are rare acid proteases found in archaea, bacteria and fungi. Certain fungi secrete acids as part of their lifestyle and interestingly these also have many eqolisin paralogs, up to nine paralogs have been recorded. This suggests a process of functional redundancy and diversification has occurred, which was the subject of the research we performed and describe here. RESULTS: We identified eqolisin homologs by means of iterative HMMER analysis of the NR database. The identified sequences were scrutinized for which new hallmarks were identified by molecular dynamics simulations of mutants in highly conserved positions, using the structure of an eqolisin that was crystallized in the presence of a transition state inhibitor. Four conserved glycines were shown to be important for functionality. A substitution of W67F is shown to be accompanied by the L105W substitution. Molecular dynamics shows that the W67 binds to the substrate via a π-π stacking and a salt bridge, the latter being stronger in a virtual W67F/L105W double mutant of the resolved structure of Scytalido-carboxyl peptidase-B (PDB ID: 2IFW). Additional problematic mutations are discussed. Upon sequence scrutiny we obtained a set of 233 sequences that was used to reconstruct a Bayesian phylogenetic tree. We identified 14 putative specificity determining positions (SDPs) of which four are explained by mere structural explanations and nine seem to correspond to functional diversification related with substrate binding and specificity. A first sub-network of SDPs is related to substrate specificity whereas the second sub-network seems to affect the dynamics of three loops that are involved in substrate binding. CONCLUSION: The eqolisins form a small superfamily of acid proteases with nevertheless many paralogs in acidic fungi. Functional redundancy has resulted in diversification related to substrate specificity and substrate binding.
Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Ácido Aspártico Endopeptidases/genética , Teorema de Bayes , Sítios de Ligação , Sequência Conservada , Evolução Molecular , Transferência Genética Horizontal/genética , Glicina/química , Modelos Moleculares , Mutação/genética , Filogenia , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources. Its maximum activity (63.7 U mL-1) was obtained in a medium containing 2% (w/v) peptone. A cultivation carried out in a 5.0 L stirred-tank bioreactor provided a maximum protease activity 9% lower than that observed in Erlenmeyer flasks, which was obtained after a significantly shorter (by 16-29%) time. Protease purification by a combination of gel-filtration chromatography resulted in a 16.9-fold increase in specific activity (248.1 U g-1). The estimated molecular weight of the purified enzyme was 50.6 kDa, and the optimal pH and temperature were 5.0 and 55 °C, respectively. The enzyme was completely inhibited by pepstatin A, and its activity enhanced by some metals. According to the inhibition profiles, it was confirmed that the purified acid protease belongs to the aspartic protease type. These results are quite promising for future development of large-scale production of such protease, which can be useful in biotechnological applications requiring high enzyme activity and stability under acidic conditions.
Assuntos
Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/isolamento & purificação , Aspergillus/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Aspergillus/química , Aspergillus/genética , Aspergillus/isolamento & purificação , Brasil , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Peso Molecular , Microbiologia do Solo , Especificidade por Substrato , TemperaturaRESUMO
Proteases ácidas pertencem a um importante grupo de enzimas industriais produzidas por fungos filamentosos, com aplicações na indústria de alimentos, de couro, farmacêutica e de cosméticos. O objetivo principal deste trabalho foi avaliar a produção de proteases ácidas extracelulares de fungos filamentosos isolados do solo do cerrado do centro-oeste brasileiro. Inicialmente, foi realizada uma triagem para avaliar a capacidade de 17 linhagens de fungos quanto à produção de protease em meio de cultura contendo Agar-leite. O fungo Aspergillus foetidus foi selecionado como melhor produtor de protease ácida extracelular. Visando à otimização da produção de proteases pelo fungo selecionado, avaliou-se a influência de diversos fatores no cultivo (pH, temperatura, agitação e diferentes fontes de nitrogênio e carbono). Após essa etapa, um planejamento experimental estatístico foi realizado com as variáveis independentes temperatura, pH inicial do meio e fonte de carbono e nitrogênio. A produção máxima de protease foi encontrada (63,7 U/mL) nas condições: pH inicial do meio igual a 7,0 a 28 ºC, 150 rpm em peptona 2% (p/v). Os estudos em biorreator demonstraram produção de protease nas condições de agitação e aeração iguais à 300 rpm e 1,0 vvm, após 120 h de cultivo. Os ensaios com diferentes temperaturas para a estimativa dos parâmetros termodinâmicos demonstraram que a protease ácida produzida pelo fungo é altamente estável apresentando máxima atividade em pH 5,0 e temperatura ótima igual a 55ºC. E, finalmente, para a purificação da enzima foi realizada cromatografia de gel-filtração. A enzima apresentou massa molecular de 50,6 kDa, e a análise do zimograma confirmou a atividade proteolítica. Além disso, a protease purificada foi inibida pelo composto pepstatina, indicando uma característica de protease ácida. Esses resultados obtidos demonstram um fungo filamentoso produtor de uma nova protease ácida com potencial aplicação para indústria farmacêutica e de cosméticos
The acid proteases belong to the most important group of industrial enzymes produced by filamentous fungi, with applications in the food, leather, pharmaceutical and cosmetics industries. This study aimed the evaluation of extracellular acid proteases production from filamentous fungi isolated from different samples of the midwestern Brazil cerrado. Initially, a screening was performed to assess the ability of the 17 strains of yeast for production of protease-agar medium containing milk culture. The Aspergillus foetidus was selected as the best producer. Aimed at optimizing the production of proteases by the selected fungus, first evaluated the influence of various factors on the cultivation (pH, temperatura, agitation and different sources of nitrogen and carbon). After this step, a statistical experimental design was carried out with the independent variables temperatura, initial pH of the medium and source of carbon and nitrogen. The best conditions for protease production were (63.7 U / mL): initial pH values greater than 7.0, at 28 °C, 150 rpm peptone 2% (w/v). Aiming future production of this protease in industrial scale, studies have shown better in bioreactor protease production under the conditions of agitation and aeration equal to 300 rpm and 1.0 vvm, after 120 h of cultive. The tests at different temperaturas to estimate the thermodynamic parameters showed that the acid protease produced by the fungus is highly stable with maximum activity at pH 5.0 and optimum temperatura of 55 °C. And finally, for the purification of the enzyme were performed gel-filtration chromatography. The enzyme had a molecular mass of 50.6 kDa, and the analysis of the zymogram showed a proteolytic band. Furthermore, the purified protease was inhibited by pepstatin compound, indicating a feature of acid protease. These results demonstrate a new filamentous fungus producing acid protease with potential application to pharmaceuticals and cosmetics
Assuntos
Peptídeo Hidrolases , Fungos , Aspergillus , Biotecnologia , Tecnologia FarmacêuticaRESUMO
The effects of glyphosate, a nonselective herbicide (1.0 or 5.0mg L-1) on digestive enzymes activity (stomach and intestine) were evaluated in juveniles of piava (Leporinus obtusidens) after 90 days of exposure. The activity of acid protease, trypsin, chymotrypsin and amylase increased with the increase of glyphosate concentration. These results indicate that glyphosate affects digestive enzyme activities in this species, and may be an indicator of poor nutrient availability when fish survive in herbicide-contaminated water.
Os efeitos do glifosato, um herbicida não seletivo (1,0 ou 5,0mg L-1), sobre a atividade de enzimas digestivas (estômago e intestino) foram avaliadas em juvenis de piava (Leporinus obtusidens) após 90 dias de exposição. A atividade da protease ácida, tripsina, quimiotripsina e amilase aumentaram com a elevação da concentração de glifosato. Esses resultados indicam que o glifosato afeta a atividade de enzimas digestivas nesta espécie e pode ser indicador da reduzida disponibilidade de nutrientes, quando peixes sobrevivem em água contaminada com este herbicida.
RESUMO
The effects of glyphosate, a nonselective herbicide (1.0 or 5.0mg L-1) on digestive enzymes activity (stomach and intestine) were evaluated in juveniles of piava (Leporinus obtusidens) after 90 days of exposure. The activity of acid protease, trypsin, chymotrypsin and amylase increased with the increase of glyphosate concentration. These results indicate that glyphosate affects digestive enzyme activities in this species, and may be an indicator of poor nutrient availability when fish survive in herbicide-contaminated water.(AU)
Os efeitos do glifosato, um herbicida não seletivo (1,0 ou 5,0mg L-1), sobre a atividade de enzimas digestivas (estômago e intestino) foram avaliadas em juvenis de piava (Leporinus obtusidens) após 90 dias de exposição. A atividade da protease ácida, tripsina, quimiotripsina e amilase aumentaram com a elevação da concentração de glifosato. Esses resultados indicam que o glifosato afeta a atividade de enzimas digestivas nesta espécie e pode ser indicador da reduzida disponibilidade de nutrientes, quando peixes sobrevivem em água contaminada com este herbicida.(AU)