Produção, isolamento e caracterização bioquímica de xilanases produzidas pelo fungo termofílico Rasamsonia emersonii por cultivo em estado sólido
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Data
2017-03-27
Autores
Zanoni, Jéssica de Araújo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O complexo enzimático xilanolítico é responsável por hidrolisar as ligações glicosídicas entre os monômeros constituintes da xilana, sendo esta um polissacarídeo presente na porção hemicelulolítica das paredes celulares vegetais, e suas enzimas se destacam pelas diversas aplicações industriais. O objetivo do projeto foi efetuar a caracterização e isolamento das xilanases produzidas pelo fungo termofílico Rasamsonia emersonii em cultivo em estado sólido. Foram realizados ensaios para avaliar os efeitos do pH, da temperatura, de diversas substâncias químicas, cátions e compostos fenólicos na atividade enzimática. A estimativa da massa molecular foi realizada por eletroforese em gel de poliacrilamida (SDS-PAGE) e por cromatografia de filtração em gel em resina Sephadex G-75. Como valor ótimo para o extrato enzimático bruto obteve-se 5,5 e 7,0 para o pH, e 80ºC para a temperatura ótima em incubação de 4 minutos. Em relação à estabilidade do extrato bruto, os maiores valores ocorreram entre as faixas de pH de 4 a 5,5, e 50ºC para temperatura. Todos os reagentes testados apresentaram diminuição da atividade enzimática sobre o extrato bruto, por outro lado, o agente quelante EDTA aumentou 4%. Os cátions testados aumentaram (Na+, K+, Cd2+, Sr2+, Ca2+) ou diminuíram (Zn2+, Al3+, Fe3+, Mn2+, Mg2+, Ni2+, Ba2+, Li+, Co2+, Cr3+ e Cu2+) a atividade. Em relação aos compostos fenólicos, somente o ácido tânico induziu decréscimo da atividade xilanolítica do extrato bruto. O pI foi estimado em 6,5 por focalização isoelétrica em gel de agarose. A massa molecular das isoformas por filtração em gel não pôde ser estimada devido a uma interação inespecífica da xilanase de maior massa molecular com a resina Sephadex. Pela análise de SDS-PAGE é possível observar que todas as isoformas se encontram abaixo de 45 kDa. Na análise por zimografia observou-se a presença de quatro xilanases, que foram isoladas pela extração do gel de poliacrilamida. Todas se mostraram mais ativas em pH 5,5 quando comparadas em pH 7,0. Avaliaram-se os parâmetros termodinâmicos da xilanase isolada C, que demonstrou mais de 6 horas à 80ºC como tempo de meia vida, e elevada resistência estrutural à desnaturação térmica quando avaliados os resultados de entropia, energia de ativação e entalpia.
The xylanolytic enzyme complex is responsible for hydrolyzing glycosidic bonds between the monomers of xylan, being a polysaccharide present of the hemicellulolitic in plant walls. These enzymes standing out because have many industrial applications. The objective of this investigation was to characterize and isolate the xylanases present in the crude extract produced by the thermophilic fungus Rasamsonia emersonii in solid state cultivation. Tests were performed to evaluate the effects of pH, temperature, various chemicals and cations on enzymatic activity, determining optimal conditions and stability of the enzymes. The molecular weight estimation was performed by polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography on Sephadex G-75 resin. Optimum pH values obtained were 5.5 and 7.0 and 80ºC for the optimum temperature for a 4 minutes incubation for the crude enzyme extract. Regarding the stability of the crude extract, the highest values occurred between the ranges of 4 to 5.5 for pH, and 50ºC for thermal stability. All the reagents tested showed enzymatic inhibition on the crude extract, on the other hand, the chelating agent EDTA increased enzyme activity by 4%. The tested cations increased (Na+ , K+ , Cd2+, Sr2+, Ca2+) or decreased (Zn2+, Al3+ , Fe3+, Mn2+, Mg2+, Ni2+, Ba2+, Li+ , Co2+, Cr3+ and Cu2+) activity. In relation to the phenolic compounds, only tannic acid induced a decrease in the xylanolytic activity of the crude extract. The pI was estimated to be around 6.5 by isoelectric focusing on agarose gel. The molecular mass of the isoforms by gel filtration could not be estimated due to a non-specific interaction of the higher molecular weight xylanase with the Sephadex gel filtration chromatography resin. By SDS-PAGE analysis it is possible to observe that all isoforms are of low molecular mass, below 45 kDa. In the electrophoretic analysis by zymography it was possible to observe the presence of four xylanases, which were isolated by extracting the enzymes from the polyacrylamide gel. All isolated xylanases were more active at pH 5.5 when compared to activities at pH 7.0. The thermodynamic parameters of the xylanase isolated C were evaluated, showing high values of half life, more than 6 hours at 80°C, and high structural resistence to thermal denaturation when the results of entropy, activation energy and enthalpy were evaluated.
The xylanolytic enzyme complex is responsible for hydrolyzing glycosidic bonds between the monomers of xylan, being a polysaccharide present of the hemicellulolitic in plant walls. These enzymes standing out because have many industrial applications. The objective of this investigation was to characterize and isolate the xylanases present in the crude extract produced by the thermophilic fungus Rasamsonia emersonii in solid state cultivation. Tests were performed to evaluate the effects of pH, temperature, various chemicals and cations on enzymatic activity, determining optimal conditions and stability of the enzymes. The molecular weight estimation was performed by polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography on Sephadex G-75 resin. Optimum pH values obtained were 5.5 and 7.0 and 80ºC for the optimum temperature for a 4 minutes incubation for the crude enzyme extract. Regarding the stability of the crude extract, the highest values occurred between the ranges of 4 to 5.5 for pH, and 50ºC for thermal stability. All the reagents tested showed enzymatic inhibition on the crude extract, on the other hand, the chelating agent EDTA increased enzyme activity by 4%. The tested cations increased (Na+ , K+ , Cd2+, Sr2+, Ca2+) or decreased (Zn2+, Al3+ , Fe3+, Mn2+, Mg2+, Ni2+, Ba2+, Li+ , Co2+, Cr3+ and Cu2+) activity. In relation to the phenolic compounds, only tannic acid induced a decrease in the xylanolytic activity of the crude extract. The pI was estimated to be around 6.5 by isoelectric focusing on agarose gel. The molecular mass of the isoforms by gel filtration could not be estimated due to a non-specific interaction of the higher molecular weight xylanase with the Sephadex gel filtration chromatography resin. By SDS-PAGE analysis it is possible to observe that all isoforms are of low molecular mass, below 45 kDa. In the electrophoretic analysis by zymography it was possible to observe the presence of four xylanases, which were isolated by extracting the enzymes from the polyacrylamide gel. All isolated xylanases were more active at pH 5.5 when compared to activities at pH 7.0. The thermodynamic parameters of the xylanase isolated C were evaluated, showing high values of half life, more than 6 hours at 80°C, and high structural resistence to thermal denaturation when the results of entropy, activation energy and enthalpy were evaluated.
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Palavras-chave
Xilanases, Rasamsonia emersonii, Caracterização bioquímica, Termodinâmica