Investigação da produção de enzimas celulolíticas em fungos filamentosos para aplicação na biodegradação de bagaço de cana-de-açúcar
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Data
2016-12-01
Autores
Kapp, Barbara Regina [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O complexo celulolítico é composto principalmente por endoglucanases, exoglucanases e β-glicosidases. As endoglucanases atuam clivando a celulose nas ligações internas da fibra, enquanto as exoglucanases hidrolisam as ligações externas. A necessidade de novas pesquisas de enzimas celulolíticas capazes de degradar eficientemente resíduos industriais como bagaço de cana-de-açúcar para diminuir o custo da hidrólise enzimática e favorecer a produção de etanol de segunda geração. Neste trabalho foram estudadas as melhores condições dos fungos filamentosos em agitação orbital, além de caracterizar os parâmetros bioquímicos como pH e temperatura ótimos e determinar o melhor produtor do complexo celulolítico e analisar a capacidade quanto à biodegradação de bagaço de cana-de-açúcar comparado com enzima comercial. Através da análise de atividade enzimática, o Aspergillus terreus mostrou-se o fungo com a maior produção nos diferentes meios de cultivo, atingindo a maior atividade enzimática no meio contendo CMC, apresentando 230,65 UI.mg proteína-1 de endoglucanase, 10,79 UI.mg proteína-1 de β-glicosidase, 57,79 UI.mg proteína-1 de exoglucanase e 68,60 UI.mg proteína-1 de atividade celulolítica total (FPAse). A análise realizada através do delineamento experimental de Plackett & Burman realizada no meio contendo glicose e celulose cristalina não mostrou influencia da composição do meio de cultura na produção das enzimas do complexo celulítico. O estudo de estabilidade ao pH revelou que o pH 3 influencia negativamente todas as enzimas do complexo celulolítico. A purificação parcial do complexo enzimático produzido por A. terreus em meio contendo CMC resultou em melhores resultados para a precipitação com acetona para β-glicosidase e exoglucanase e FPAse e que a enzima exoglucanase fica retida na membrana de diálise. O peso molecular do complexo enzimático purificado parcialmente produzido por A. terreus em meio contendo CMC é de 73 kDa. A conversão de celulose em glicose do complexo produzido por A. terreus purificado parcialmente foi inferior à enzima comercial sobre bagaço de cana pré-tratado com sulfito alcalino, porém, com conversão superior à enzima comercial no bagaço de cana in natura nos tempos de 4 e 8 horas de incubação.
The cellulolytic complex is composed mainly of endoglucanases, exoglucanases and β-glycosidases. Endoglucanases act by cleaving the cellulose in the internal bonds of the fiber, while the exoglucanases hydrolyze the outer bonds. The need for new research on cellulolytic enzymes capable of efficiently degrading industrial residues such as sugarcane bagasse to reduce the cost of enzymatic hydrolysis and favor the production of second generation ethanol. In this work the best conditions of the filamentous fungi were studied in orbital agitation, besides characterizing the biochemical parameters as pH and temperature optimum and to determine the best producer of the cellulolítico complex and to analyze the capacity regarding the biodegradation of sugarcane bagasse compared to Commercial enzyme. Through enzymatic activity analysis, Aspergillus terreus showed the fungus with the largest output in the different culture media, achieving higher production in D medium, showing 230,65 65 μmol.min-1.mg protein-1 for endoglucanase, 10788,34 nmol.min-1.mg protein-1 for β-glycosidase, 57.79 μmol.min-1.mg protein-1 for exoglucanase and 68,60 μmol.min-1.mg protein-1 for Total Cellulolytic Activity – Filter Papel Activity (FPAse). The analysis made with the Plackett & Burman design for the medium content glucose and crystal cellulose showed that the medium composition not show an influence on the enzyme production of cellulolytic complex. The enzymatic stability of pH showed that pH 3 has a negative influence for all enzymatic complex. The endoglucanase and FPAse tend to 0% of residual activity after 24 hours of incubation in all studied pHs for all fungi. The partial purification showed best results with the acetone precipitation and, also, the exoglucanase enzyme was the only one retained on the dialysis membrane. The enzymatic complex produced by A. terreus in medium content CMC has a molecular weight of 73 kDa. The conversion of cellulose to glucose from the partially purified A. terreus complex was lower than the commercial enzyme on alkali sulfite pretreated sugarcane bagasse, but with conversion higher than the commercial enzyme in the cane bag in natura at times of 4 and 8 hours incubation.
The cellulolytic complex is composed mainly of endoglucanases, exoglucanases and β-glycosidases. Endoglucanases act by cleaving the cellulose in the internal bonds of the fiber, while the exoglucanases hydrolyze the outer bonds. The need for new research on cellulolytic enzymes capable of efficiently degrading industrial residues such as sugarcane bagasse to reduce the cost of enzymatic hydrolysis and favor the production of second generation ethanol. In this work the best conditions of the filamentous fungi were studied in orbital agitation, besides characterizing the biochemical parameters as pH and temperature optimum and to determine the best producer of the cellulolítico complex and to analyze the capacity regarding the biodegradation of sugarcane bagasse compared to Commercial enzyme. Through enzymatic activity analysis, Aspergillus terreus showed the fungus with the largest output in the different culture media, achieving higher production in D medium, showing 230,65 65 μmol.min-1.mg protein-1 for endoglucanase, 10788,34 nmol.min-1.mg protein-1 for β-glycosidase, 57.79 μmol.min-1.mg protein-1 for exoglucanase and 68,60 μmol.min-1.mg protein-1 for Total Cellulolytic Activity – Filter Papel Activity (FPAse). The analysis made with the Plackett & Burman design for the medium content glucose and crystal cellulose showed that the medium composition not show an influence on the enzyme production of cellulolytic complex. The enzymatic stability of pH showed that pH 3 has a negative influence for all enzymatic complex. The endoglucanase and FPAse tend to 0% of residual activity after 24 hours of incubation in all studied pHs for all fungi. The partial purification showed best results with the acetone precipitation and, also, the exoglucanase enzyme was the only one retained on the dialysis membrane. The enzymatic complex produced by A. terreus in medium content CMC has a molecular weight of 73 kDa. The conversion of cellulose to glucose from the partially purified A. terreus complex was lower than the commercial enzyme on alkali sulfite pretreated sugarcane bagasse, but with conversion higher than the commercial enzyme in the cane bag in natura at times of 4 and 8 hours incubation.
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Palavras-chave
Fungos filamentosos, Enzimas, Enzimas celulolíticas, Aspergillus terréus, Bioconversão, Bagaço de cana, Filamentous fungi, Enzymes, Cellulolytic enzymes, Aspergillus terreus, Bioconversion, Sugar cane bagasse