Orientador: Prof. Dr. Wanius José Garcia da Silva / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2016. / A hidrolise enzimatica da celulose e realizada pela acao sinergica de pelo menos tres tipos de celulases distintas: endoglucanases, exoglucanases e ¿À.glicosidases. As endoglucanases e celobiohidrolases sao frequentemente inibidas pelo aumento da concentracao de celobiose (dimero de glicose) no meio reacional. As ¿À-glicosidases sao enzimas que clivam celobiose em monomeros de glicose. Portanto, as ¿À-glicosidases sao essenciais ao processo de hidrolise da celulose por impedirem o acumulo de celobiose e, assim, evitar a diminuicao da taxa de hidrolise. Processos de pre-tratamento da biomassa lignocelulosica sao empregados, antes da reacao de hidrolise enzimatica da celulose, a fim de retirar a fracao de lignina e aumentar a taxa de conversao da celulose em glicose. Porem, estes processos de pre-tratamento da biomassa lignocelulosica nao sao 100% eficientes na remocao da lignina. Estudos previos mostraram que a adicao de lignina a celulose pura pode causar a reducao da liberacao de acucar em valores superiores a 60%. Assim, neste estudo, nos caracterizamos a adsorcao nao produtiva da enzima ¿À-glicosidase da familia GH1 da bacteria Thermotoga petrophila (TpBGL1) em ligninas extraidas de diferentes biomassas (cana-de-acucar e eucalipto). Em pH 7 e 6, nossos resultados indicaram que a repulsao eletrostatica enfraquece a adsorcao nao produtiva de TpBGL1 em ligninas. Contudo, em pH 4 a atracao eletrostatica fortalece a adsorcao nao produtiva. Alem disso, o aumento da temperatura de 25 oC para 70 oC nao resultou em um aumento significativo da adsorcao de TpBGL1 em ligninas, provavelmente porque nao ocorre um aumento significativo de regioes hidrofobicas na estrutura da enzima expostas ao solvente. Todas as informacoes obtidas neste estudo poderao ser uteis para aplicacoes biotecnologicas no campo de conversao de polissacarideos estruturais em bioenergia. / The enzymatic hydrolysis of cellulose to glucose requires at least three types of enzymes working synergistically: endoglucanases that randomly cleave the â-1,4-glycosidic linkages of cellulose, cellobiohydrolases which cleave off cellobiose units from the reducing or nonreducing end of cellulose chain, and â-glucosidases responsible for hydrolysis of the released cellobiose to glucose. Endoglucanases and cellobiohydrolases are normally inhibited by cellobiose, therefore, â-glucosidases are essential to avoid the decrease the hydrolysis rates of cellulose over time due to cellobiose accumulation. The presence of residual lignin after pretreatments may affect negatively the enzymatic hydrolysis of cellulose to glucose. Cellulases bind to lignin, deactivating the enzymes and reducing the overall enzymatic activities. In this study, we examined the non-productive adsorption of one â-glucosidase from Thermotoga petrophila, belonging to the family GH1, on the lignin preparations from both sugarcane (grasses) and eucalyptus (hardwood). GH1 â-glucosidase adsorption onto lignins was found to be strongly pH-dependent, suggesting that the adsorption is electrostatically modulated. At pH 7 and 6, electrostatic repulsion weakens the non-productive adsorption of GH1 â-glucosidase to lignins. However, at pH 4, attractive electrostatic interactions strengthen the non-productive adsorption of GH1 â-glucosidase to lignins. Finally, the increase of temperature did not result in the increase of GH1 â-glucosidase adsorption, probably because there is no significant increase in hydrophobic regions in the GH1 â-glucosidase structure. These studies can be useful in the field of plant structural polysaccharides conversion into bioenergy.

An investigation was made into the isolation and screening of highly cellulolytic anaerobic fungi and their production of cellulolytic enzymes using immobilised rhizomycelia. A total of 46 anaerobic fungi were isolated on cellulosic substrates from ruminant and non-ruminant herbivores. Primary screening of these isolates was performed using dye release from cellulose-azure which qualitatively detected cellulolytic activity. Twelve isolates were chosen on the basis of their maximum solubilisation rates of the labelled cellulose and then subjected to secondary screening which involved the quantification of enzyme activity. The enzyme mixtures were characterised by carboxymethylcellulase, xylanase, B-glucosidase, B-xylosidase and cellobiase assays, measured by the production of either reducing sugars, p-nitrophenol or glucose. All strains produced a number of enzymes that allowed them to hydrolyse straw and highest enzyme activity was measured in static cultures grown on 0.5% straw. A monocentric isolate, Piromyces strain KSX1 from a red kangaroo, and a cattle polycentric isolate, Orpinomyces strain 478P1, were selected for study of cellulolytic enzyme production on the basis of high fibre digestion capability and amenability toward encapsulation. The immobilised polycentric strain proved to be operationally superior to strain KSX1 as strain 478P1 did not produce any viable growth in the culture liquor. Studies into single batch cultures of free cells of strains KSX1 and 478P1 revealed that the maximum specific rate of B-glucosidase production occurred concomitantly with maximum specific growth rate suggesting that the immobilised fungus must grow for continuous enzyme production to occur. Although the physiology of cellulase synthesis in strains KSX1 and 478P1 was found to be growth-associated, immobilisation of the fungus offered the advantage of the repeat-batch use of cells with the accumulation of extracellular enzymes after each batch. Thus, operational gains were the key issues in assessing the potential application of immobilised anaerobic fungi in the production of cellulolytic enzymes. The repeat-batch system was operationally more efficient than the free cell batch cultures because immobilisation removed the need of reculturing the cells for every single batch. / Doctor of Philosophy (PhD)