Caracterização de uma endoglucanase termoestável do fungo termofílico Rasamsonia emersonii S10
Carregando...
Data
2016-07-08
Autores
Chierotti, Maria Cecilia Maia [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Um dos interesses biotecnológicos e econômicos da atualidade é otimizar o emprego da biomassa em processos industriais mediado por biocatalisadores, como as enzimas. O objetivo do presente trabalho foi purificar e caracterizar uma endoglucanase produzida pelo fungo Rasamsonia emersonii S10, em fermentação em estado sólido, tendo como substrato resíduos lignocelulósicos. A partir de solução enzimática bruta detectou-se 2 isoformas da enzima, com massas moleculares de 45 e 60 kDa, sendo escolhida a primeira para ser purificada. A solução foi clarificada em carvão ativado e concentrada por ultrafiltração tangencial. A endoglucanase foi purificada em coluna cromatográfica iônica e de exclusão molecular, intermediada por diálise. O processo levou a um fator de purificação de 6,7 e rendimento de 8,8%. A enzima apresentou temperatura ótima de 85,3 ºC e pH 4,0. Ativação foi evidenciada quando a enzima foi mantida de 40 até 60 ºC. A enzima foi estável, quando mantida em temperaturas de 65 a 80 ºC e faixa de pH entre 6 e 7,5. Estudo do efeito de íons e compostos orgânicos revelaram ativação em presença de MnCl2 e inibição por HgCl2 e ácido 4-hidroxibenzóico e 5-hidroximetilfurfural. A enzima mostrou afinidade por CMC, porém também foi capaz de hidrolisar Avicel®. A CMCase exibiu Km de 3,53 mg.mL-1 e Vmax de 2,86 µmol/min.mL. Os parâmetros termodinâmicos indicaram uma endoglucanase estável a altas temperaturas, com tempo de meia vida de 355 min a 40 ºC e temperatura de fusão em 89 ºC.
Nowadays, one of the technological and economic interests is to optimize the use of biomass in industrial processes mediated by biocatalysts such as enzymes. The objective of this study was to purify and characterize an endoglucanase produced by the fungus Rasamsonia emersonii S10 in solid state fermentation, as substrate lignocellulosic residues. From the crude enzyme solution was detected 2 isoform of the enzyme, with a molecular weight of 45 kDa and 60, the first being chosen to be purified. The solution was clarified by activated carbon and concentrated by tangential ultrafiltration. The endoglucanase was purified by ion chromatographic and molecular exclusion mediated by dialysis. The process led to a purification factor of 6.7 and 8.8% yield. The enzyme showed optimum temperature of 85.3 °C and pH 4.0. Activation was observed when the enzyme was maintained at 40 to 60 °C. The enzyme was stable when maintained at temperatures 65-80 °C and pH between 6 and 7.5. Study of the effect of ions and organic compounds showed activation in the presence of MnCl2 and HgCl2 inhibition and 4- hydroxybenzoic acid and 5-hydroxymethylfurfural. The enzyme showed affinity for CMC, but was also able to hydrolyze Avicel®. The CMCase exhibited Km 3.53 mg.mL-1 and Vmax of 2.86 mmol/min.mL-1 . The thermodynamic parameters indicated endoglucanase stable at high temperatures, with half-life of 355 min at 40 °C and the melting temperature at 89 °C.
Nowadays, one of the technological and economic interests is to optimize the use of biomass in industrial processes mediated by biocatalysts such as enzymes. The objective of this study was to purify and characterize an endoglucanase produced by the fungus Rasamsonia emersonii S10 in solid state fermentation, as substrate lignocellulosic residues. From the crude enzyme solution was detected 2 isoform of the enzyme, with a molecular weight of 45 kDa and 60, the first being chosen to be purified. The solution was clarified by activated carbon and concentrated by tangential ultrafiltration. The endoglucanase was purified by ion chromatographic and molecular exclusion mediated by dialysis. The process led to a purification factor of 6.7 and 8.8% yield. The enzyme showed optimum temperature of 85.3 °C and pH 4.0. Activation was observed when the enzyme was maintained at 40 to 60 °C. The enzyme was stable when maintained at temperatures 65-80 °C and pH between 6 and 7.5. Study of the effect of ions and organic compounds showed activation in the presence of MnCl2 and HgCl2 inhibition and 4- hydroxybenzoic acid and 5-hydroxymethylfurfural. The enzyme showed affinity for CMC, but was also able to hydrolyze Avicel®. The CMCase exhibited Km 3.53 mg.mL-1 and Vmax of 2.86 mmol/min.mL-1 . The thermodynamic parameters indicated endoglucanase stable at high temperatures, with half-life of 355 min at 40 °C and the melting temperature at 89 °C.
Descrição
Palavras-chave
Celulase, Endoglucanase, Rasamsonia emersonii, Termoestabilidade, Thermostability