Complexo celulolítico e hemicelulolítico do fungo endofítico Fusarium verticillioides e sua aplicação para sacarificação do bagaço de cana.

• Autor(a) principal: Almeida, Maíra Nicolau de
• Data de Publicação: 2013
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/321
• topic: Celulase; Hemicelulase; Fusarium verticillioides; Cellulase; Hemicellulase; Fusarium verticillioides; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

In this work, the endophytic fungus Fusarium verticillioides was evaluated in relation to its potential to produce cellulases and hemicellulases aiming to its application on biomass saccharification for ethanol production. The culture medium used for cellulase and xylanase production was optimized. Optimum levels of carbon source concentration (corn straw), nitrogen source concentration (sodium nitrate) and time of cultivation were obtained. Endoglucanase and xylanase activities increased from 2.8 U/mL to 8.0 U/mL and from 3.4 U/mL to 114.0 U/mL, respectively. The optimal pH and temperature were determined for endoglucanase (5.6, 80 °C), cellobiase (5.6, 60 °C), FPase (6.0, 55 °C) and xylanase (7.0, 50 °C). The optimized crude extract was applied in saccharification and fermentation of sugarcane bagasse from which 9.7 g/L of ethanol was produced at an ethanol/biomass (g/g) yield of 0.19. A second cultivation medium containing different nitrogen sources (urea, yeast extract, peptone and ammonium sulfate) and forage as carbon source was established throught Plackett Burman design for cellulases production. Two contrasting media, M1 and M2 were defined for higher endoglucanase and cellobiase production, respectively. Mixtures of these two extracts were tested on a sugarcane bagasse saccharification and extract M1 presented the best performance, yielding 26.6 % of glucan conversion and 40.4 % of xylan conversion. Different dosages of protein (10 mg/g, 20 mg/g and 40 mg/g of dry biomass) were tested intending to improve the hydrolysis performance of M1 extract. Saccharification rates of 43.4 % and 73.1 % were observed for glucan and xylan fractions respectively when 40 mg/g was used. A high protein adsorption on substrate was observed during saccharification assays employing M1 extract and that can explain de lower sugar productivity in the saccharification process along the time. The fungus F. verticillioides and the fungus Acremonium zeae were evaluated in relation to its abilities to ferment simple sugars and lignocellulosic biomass. The fungus Fusarium verticillioides produced ethanol from glucose, xylose and a mixture of these two sugars in limited oxygen conditions with yields of 0.47 g/g, 0.46 g/g and 0.50 g/g of ethanol per sugar utilized. The fungus Acremonium zeae produced ethanol from glucose, xylose and mixture of these two sugars with yields of 0.37 g/g, 0.39 g/g and 0.48 g/g of ethanol per sugar utilized. Both fungi were able to co-ferment glucose and xylose. Fusarium verticillioides and A. zeae produced high endoglucanase and xylanase activities using sugarcane bagasse as substrate. Ethanol production from 40 g/L of pre-treated sugarcane bagasse was 4.6 g/L and 3.9 g/L for Fusarium verticillioides and A. zeae, respectively. Both fungi showed features suitable for consolidated bioprocessing. Great interest was generated by the biochemical characteristics of the endoglucanase activity, as activity in high temperatures and stability in various pHs. Then it was developed a purification and characterization study of endoglucanase activity from the fungus F. verticillioides. A multienzyme complex, E1C, and a free endoglucanase, E2 were purified. The E1C contained two endoglucanases (GH6 and GH10), one cellobiohydrolase (GH7) and one xylanase (GH10). The temperature of maximal activity was 80 °C for both E1C and E2. The free enzyme and the complex were very thermostable at 50 °C and 60 °C. The activation energies for E1C and E2 were 21.3 KJ/mol and 27.5 KJ/mol, respectively. Maximum activity of E1C was encountered at pH 4.5 and of E2 was at pH 5.5; E1C presented high stability after 24 h of pre-incubation at pH ranging from 2.6 to 8.0. The KM value for E1C was 10.25 g/L while for E2 was 6.58 g/L using carboxymethylcellulose as substrate. Both E1C and E2 were significantly activated by Mn2+, CoCl2, furfural, hydroxymethylfurfural and dithiothreitol while they were inhibited by SDS, CuSO4, FeCl3, AgNO4, ZnSO4 and HgCl2. Both E1C and E2 presented higher activity towards barley-β-glucan than carboxymethylcellulose (CMC), indicating endo-β-1,3-1,4-glucanase activity. The enzymes were able to hydrolyze cellopentaose, cellotetraose and cellotriose, however, in different mode of action. In all the assays evaluating stability and efficiency E1C showed better performance than E2, suggesting advantages generated by the physical interaction between proteins.