β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar

• Autor(a) principal: Andrade, Lorena Gusmão Alvarenga de
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2475
• topic: Fungo; Chrysoporthe cubensis; β-Glicosidases; Bagaço de cana; Purificação; Fungus; Chrysoporthe cubensis; β-glucosidases; Bagasse; Purification; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

In this work, a β-glucosidases complex from the phytopathogen fungus Chrysoporthe cubensis LPF-1 was purified, biochemistry and kinetically characterized and evaluated for its potential application in the process of saccharification of sugarcane bagasse. C. cubensis was grown in solid state fermentation (SSF) contaning wheat bran as carbon source and after five days of fermentation the enzymatic extract was obtaneid. A Ec complex containing three β-glucosidases was purified by ion exchange and gel filtration chromatographies and the end of the process the Ec showed a purification factor of 2.05 times with a yield of 64.5%. The molecular weight of the β-glucosidases was as 85,43 and 74,17 kDa for E2 and E3, respectively, and for E1a, E1b, E1c was 63,09, 60,25 e 56,23 kDa, respectively, when estimated by SDS-PAGE. The complex of β-glucosidases (Ec) exhibited maximum activity at pH 4.0 and 60 ° C. Ec was stable in the pH range of 2.0- 8.0 and showed high thermal stability at temperatures of 50, 60 and 70 ° C. The Ec half- life values were 52 hours and 39 minutes, 35 hours and 30 minutes and 15.3 minutes at 50, 60 and 70 ° C, respectively. The Ec of β-glucosidase activity was completely innibited by HgCl2 and MnSO4 at a concentration of 10 mM and the complex of β- glucosidases was specific for glucose residues in β position. The KM values for Ec of 0.132 and 0.7816 mM and Vmax of 0.416 and 0.3414 μM.min-1 were obtained using the ρNPβGlc and cellobiose substrates, respectively. The Ec showed competitive inhibition of glucose to type with Ki value of 8.43 mM. The β-glucosidases were employed in saccharification of sugarcane bagasse, previously submitted to the alkaline pretreatment. Ec was used as a source of supplementation for both extract from C. cubensis as well as in a commercial cellulase cocktail. The saccharification results with the supplemented preparations with β-glucosidases were compared with those obtained employing a commercial cocktail and only the extract of C. cubensis and the production of glucose and xylose were evaluated for 72 hours of hydrolysis. Saccharification performed with commercial cocktail supplemented with the β-glucosidases promoted the release of 2.82 g / L of glucose and 0.98 g / L xylose whereas only with the commercial cocktail were released 1.78 g / L of glucose and 0.85 g / L of xylose. On the other hand, the saccharification with the C. cubensis extract produced 1.69 g / L of glucose and 1.64 g / L xylose while the same fraction supplemented with β-glucosidases promoted the release of 2.62 g / L of glucose and 2.65 g / L of xylose. Thus, the results obtained suggests that the β-glucosidases of C. cubensis evaluated in this work have potential to be used as a source of supplemental other enzyme preparations, since the hydrolysis efficiency was higher than that observed in commercial cocktail of cellulase as well as extract of C. cubensis.