Produção de enzimas despolimerizantes de material vegetal pelo fungo termofílico Rasamsonia emersonii S10 por cultivo em estado sólido
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Data
2018-08-02
Autores
Rosa, Isabel Zaparoli
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Universidade Estadual Paulista (Unesp)
Resumo
Os fungos termofílicos verdadeiros fazem parte de um grupo ainda não muito conhecido, sendo relativamente poucas as espécies descritas. Muitas das cepas consideradas termofílicas são na verdade termotolerantes, ou seja, apresentam maior crescimento em temperaturas mesofílicas, mas toleram temperaturas mais elevadas. O fungo Rasamsonia emersonii S10, recentemente isolado, apresenta um perfil termofílico verdadeiro, pouco comum, com crescimento em temperaturas ao redor de 60 °C. O presente trabalho buscou conhecer mais sobre as características de crescimento desse fungo e das enzimas extracelulares por ele secretadas quando em cultivos em substratos sólidos lignocelulósicos. Entre as enzimas testadas, endoglucanase, exoglucanase, β-glicosidase e β-xilosidase foram as que foram detectadas com as maiores atividades. Foram selecionadas as enzimas β-glicosidase e βxilosidase para estudos subsequentes, com cultivo do fungo em diferentes substratos, seguindo-se da purificação de ambas as enzimas e caracterização da β-glicosidase. As proteínas foram purificadas em coluna cromatográfica de troca aniônica. Ao final do processo, chegou-se a um fator de purificação de 11 e rendimento de 96% para a β-glicosidase. Estudos bioquímicos revelaram que as condições melhores para atividade da enzima foram pH 3,5 e temperatura de 70 °C. Essa enzima, quando em ausência de substrato, foi estável em estreita faixa de pH (5 a 8,5) e em temperaturas entre 45 a 75 °C. Os parâmetros cinéticos Km e Vmax obtidos através de ajuste não linear da curva foram similares para os substratos pNPG e pNPGal, sendo eles de 1,95 e 2,54 µmol/min.mL-1 e 31,53 e 31,42 mg/mL-1, respectivamente, e os valores calculados de kcat e da eficiência catalítica (kcat/km) foram respectivamente, de 163,63 e 102,79 s-1 e 83,77 e 40,46 mM-1s-1. A β-glicosidase purificada foi suscetível a todos os cátions, sendo que sua atividade residual foi mantida apenas até 25% em presença de Cd2+, Hg2+e K+, chegando a 5% e 0 em presença de Ag+ e Cu+2. As análises termodinâmicas indicaram que a 70 °C a enzima apresenta meia vida de 7 horas e necessita de 23,50 horas para redução de sua atividade a 10% do valor inicial.
True thermophilic fungi are part of a group not yet well known, with few species described. Many of the strains considered thermophilics are, in fact, thermotolerants. Fungus Rasamsonia emersonii S10, recently isolated by the group of the Laboratory of Biochemistry and Applied Microbiology, presented an uncommon thermophilic profile, with a higher growth in temperatures around 60 °C. Due to the fact that this growth profile is rare among fungal species, the present project sought to know more about the growth characteristics of this fungus and extracellular enzymes secreted in solid cultures by using lignocellulosic residues as substrates. Endoglucanase, exoglucanase, βglycosidase and β-xylosidase demonstrated the best quantitative results among the enzymes tested. For the following steps, β-glucosidase and β-xylosidase enzymes were selected. Quantitative analysis was carried out with the enzymes, ending with the purification of both and β-glycosidase characterization. The enzymes were purified on anion exchange chromatographic column. The process led to a purification factor of 11 and yield of 96% for β-glucosidase. The biochemical data revealed optimum conditions for enzyme activity at pH 3.5 and temperature of 70 °C and stability profiles in the absence of substrate with pH from 5 to 8.5 and temperature from 45 to 75 °C. The kinetic parameters Km and Vmax obtained by non-linear curve fitting were similar for the substrates pNPG and pNPGal, being 1.95 and 2.54 μmol/min.mL-1 and 31.53 and 31.42 mg/ml-1, respectively, and the calculated values of kcat and the catalytic efficiency (kcat/km) were, respectively, 163.63 and 102.79 s-1 and 83.77 and 40.46 mM -1s -1. The purified β-glycosidase was susceptible to all cations, and its residual activity was maintained only up to 25% in presence of Cd2+, Hg2+ and K+, reaching 5% and 0 in the presence of Ag+ and Cu+2. The thermodynamic analysis indicated that at 70° C the enzyme had a half-life of 7 hours and required 23.5 hours to reduce its activity to 10% of the initial value.
True thermophilic fungi are part of a group not yet well known, with few species described. Many of the strains considered thermophilics are, in fact, thermotolerants. Fungus Rasamsonia emersonii S10, recently isolated by the group of the Laboratory of Biochemistry and Applied Microbiology, presented an uncommon thermophilic profile, with a higher growth in temperatures around 60 °C. Due to the fact that this growth profile is rare among fungal species, the present project sought to know more about the growth characteristics of this fungus and extracellular enzymes secreted in solid cultures by using lignocellulosic residues as substrates. Endoglucanase, exoglucanase, βglycosidase and β-xylosidase demonstrated the best quantitative results among the enzymes tested. For the following steps, β-glucosidase and β-xylosidase enzymes were selected. Quantitative analysis was carried out with the enzymes, ending with the purification of both and β-glycosidase characterization. The enzymes were purified on anion exchange chromatographic column. The process led to a purification factor of 11 and yield of 96% for β-glucosidase. The biochemical data revealed optimum conditions for enzyme activity at pH 3.5 and temperature of 70 °C and stability profiles in the absence of substrate with pH from 5 to 8.5 and temperature from 45 to 75 °C. The kinetic parameters Km and Vmax obtained by non-linear curve fitting were similar for the substrates pNPG and pNPGal, being 1.95 and 2.54 μmol/min.mL-1 and 31.53 and 31.42 mg/ml-1, respectively, and the calculated values of kcat and the catalytic efficiency (kcat/km) were, respectively, 163.63 and 102.79 s-1 and 83.77 and 40.46 mM -1s -1. The purified β-glycosidase was susceptible to all cations, and its residual activity was maintained only up to 25% in presence of Cd2+, Hg2+ and K+, reaching 5% and 0 in the presence of Ag+ and Cu+2. The thermodynamic analysis indicated that at 70° C the enzyme had a half-life of 7 hours and required 23.5 hours to reduce its activity to 10% of the initial value.
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Palavras-chave
Fungo termofílico, Celulases, Rasamsonia emersonii, Thermophilic fungus, Cellulases