Celulases de fungos endofíticos: estudo da produção por fermentação submersa e aplicação na sacarificação da palha de cana-de-açúcar
Carregando...
Data
2018-07-30
Autores
Tartarine, Naiani
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Celulases microbianas têm sido empregadas na sacarificação de materiais lignocelulósicos para a obtenção de glicose, visando a produção de etanol celulósico. Entretanto, o custo dessas enzimas encarece o processo, e por isso a produção das mesmas a partir de fungos filamentosos cultivados em materiais lignocelulósicos, substratos de baixo custo, amplamente disponíveis e indutores da síntese enzimática, torna-se uma boa opção. Neste trabalho, com o objetivo de melhorar a produção de celulases através de metodologias de planejamento de experimentos, foram utilizados dois fungos endofíticos, cultivados por fermentação submersa (FSbm), usando como substratos o farelo de soja e a palha de cana de açúcar pré-tratada hidrotermicamente, cedida pela empresa GranBio, em cultivos isolados e co-cultivos, agitados. As atividades máximas obtidas nos extratos enzimáticos produzidos por planejamento experimental foram de 3,77 U/mL e 3,66 U/mL de endoglucanase, e 18,8 U/mL e 17,5 U/mL de β-glicosidase, no co-cultivo dos fungos Botryosphaeria sp. AM01 e Saccharicola sp. EJC04, e no cultivo isolado de Botryosphaeria sp. AM01, respectivamente. As condições ótimas de atividade das enzimas endoglucanase e β-glicosidase do melhor extrato foram pH 5,5 e 60 ºC. Estas enzimas foram estáveis quando incubadas por 24 horas em pH 5,0 e 5,5, e menos de 20% da atividade de ambas foi perdida entre 35 e 55 ºC. Os ensaios de inibição por glicose mostraram que a β-glicosidase tem sua atividade reduzida proporcionalmente ao fornecimento deste açúcar, porém, mesmo com a máxima quantidade de glicose fornecida (12 mmol/L), a atividade relativa mostrou-se mediana (64 %). Em relação à influência do etanol, a presença de 5 e 10% deste álcool aumentou em 4 e 5 %, respectivamente, a atividade enzimática de β-glicosidase, ocorrendo inibição em concentrações superiores. Em relação à sacarificação das palhas de cana, as maiores concentrações de glicose foram obtidas quando utilizaram-se a palha submetida a pré-tratamento hidrotérmico ácido seguido de alcalino (3,29 mg/mL) e a palha pré-tratada hidrotermicamente cedida pela empresa GranBio (2,66 mg/mL).
Microbial cellulases have been used in the saccharification of lignocellulosic materials to obtain glucose, aiming to produce cellulosic ethanol. However, the cost of these enzymes makes the process expensive, so their production from filamentous fungi grown in lignocellulosic materials, low-cost and widely available substrates and inducers of enzymatic synthesis, is a good option. In order to improve cellulase production through experiment planning methodologies, two endophytic fungi, were cultivated by submerged fermentation (FSbm). Soybean meal and hydrothermally pretreated sugarcane straw, provided by GranBio, were used as substrate, in agitated isolated cultures and co-cultivations. The maximal activities obtained in the enzymatic extracts produced by the experimental design were 3.77 U/mL and 3.66 U/mL endoglucanase and 18.8 U/mL and 17.5 U/mL β-glycosidase in the co-cultivation of the fungi Botryosphaeria sp. AM01 and Saccharicola sp. EJC04, and in the isolated cultivation of Botryosphaeria sp. AM01, respectively.. The optimal activity conditions of the endoglucanase and β-glucosidase enzymes from the best selected extract were pH 5.5 and 60 ° C. These enzymes were stable when incubated for 24 hours at pH 5.0 and 5.5, and less than 20% of the activity of both of them was lost at 35-55 °C. The glucose-inhibition assays showed that β-glucosidase activity was reduced proportionally to the supply of this sugar, but even with the maximum amount of glucose supply (12 mmol/L), the relative activity was shown to be median (64 %). Regarding the influence of ethanol, the presence of 5 and 10% of this alcohol increased in 4 and 5%, respectively, the enzymatic activity of β-glucosidase, occurring inhibition in higher concentrations. Regarding the saccharification of sugarcane straw, the highest glucose concentrations were obtained with the straws that were submitted to the acid hydrothermal treatment followed by the alkaline treatment (3.29 mg/mL) and the hydrothermally pretreated straw provided by the company GranBio (2.66 mg/mL).
Microbial cellulases have been used in the saccharification of lignocellulosic materials to obtain glucose, aiming to produce cellulosic ethanol. However, the cost of these enzymes makes the process expensive, so their production from filamentous fungi grown in lignocellulosic materials, low-cost and widely available substrates and inducers of enzymatic synthesis, is a good option. In order to improve cellulase production through experiment planning methodologies, two endophytic fungi, were cultivated by submerged fermentation (FSbm). Soybean meal and hydrothermally pretreated sugarcane straw, provided by GranBio, were used as substrate, in agitated isolated cultures and co-cultivations. The maximal activities obtained in the enzymatic extracts produced by the experimental design were 3.77 U/mL and 3.66 U/mL endoglucanase and 18.8 U/mL and 17.5 U/mL β-glycosidase in the co-cultivation of the fungi Botryosphaeria sp. AM01 and Saccharicola sp. EJC04, and in the isolated cultivation of Botryosphaeria sp. AM01, respectively.. The optimal activity conditions of the endoglucanase and β-glucosidase enzymes from the best selected extract were pH 5.5 and 60 ° C. These enzymes were stable when incubated for 24 hours at pH 5.0 and 5.5, and less than 20% of the activity of both of them was lost at 35-55 °C. The glucose-inhibition assays showed that β-glucosidase activity was reduced proportionally to the supply of this sugar, but even with the maximum amount of glucose supply (12 mmol/L), the relative activity was shown to be median (64 %). Regarding the influence of ethanol, the presence of 5 and 10% of this alcohol increased in 4 and 5%, respectively, the enzymatic activity of β-glucosidase, occurring inhibition in higher concentrations. Regarding the saccharification of sugarcane straw, the highest glucose concentrations were obtained with the straws that were submitted to the acid hydrothermal treatment followed by the alkaline treatment (3.29 mg/mL) and the hydrothermally pretreated straw provided by the company GranBio (2.66 mg/mL).
Descrição
Palavras-chave
Celulase, Fungos endofíticos, Fermentação, Lignocelulose, Hidrólise