<p>The hydrolysis kinetics of bacterial cellulose and its derivatives by <i>Trichoderma reesei</i> cellulases was studied. The cellulose surface erosion model was introduced to explain the gradual and strong retardation of the rate of enzymatic hydrolysis of cellulose. This model identifies the decrease in apparent processivity of cellobiohydrolases during the hydrolysis as a major contributor to the decreased rates. Both enzyme-related (non-productive binding) and substrate-related (erosion of cellulose surface) processes contribute to the decrease in apparent processivity. Furthermore, the surface erosion model allows, in addition to conventional endo-exo synergism, the possibility for different modes of synergistic action between cellulases. The second mode of synergism operates in parallel with the conventional one and was found to be predominant in the hydrolysis of more crystalline celluloses and also in the synergistic action of two cellobiohydrolases. </p><p>A mechanism of substrate inhibition in synergistic hydrolysis of bacterial cellulose was proposed whereby the inhibition is a result of surface dilution of reaction components (bound cellobiohydrolase and cellulose chain ends) at lower enzyme-to-substrate ratios. </p><p>The inhibition of cellulases by the hydrolysis product, cellobiose, was found to be strongly dependent on the nature of the substrate. The hydrolysis of a low molecular weight model substrate, such as para-nitrophenyl cellobioside, by cellobiohydrolase I is strongly inhibited by cellobiose with a competitive inhibition constant around 20 μM, whereas the hydrolysis of cellulose is more resistant to inhibition with an apparent inhibition constant around 1.5 mM for cellobiose.</p>

The hydrolysis kinetics of bacterial cellulose and its derivatives by Trichoderma reesei cellulases was studied. The cellulose surface erosion model was introduced to explain the gradual and strong retardation of the rate of enzymatic hydrolysis of cellulose. This model identifies the decrease in apparent processivity of cellobiohydrolases during the hydrolysis as a major contributor to the decreased rates. Both enzyme-related (non-productive binding) and substrate-related (erosion of cellulose surface) processes contribute to the decrease in apparent processivity. Furthermore, the surface erosion model allows, in addition to conventional endo-exo synergism, the possibility for different modes of synergistic action between cellulases. The second mode of synergism operates in parallel with the conventional one and was found to be predominant in the hydrolysis of more crystalline celluloses and also in the synergistic action of two cellobiohydrolases. A mechanism of substrate inhibition in synergistic hydrolysis of bacterial cellulose was proposed whereby the inhibition is a result of surface dilution of reaction components (bound cellobiohydrolase and cellulose chain ends) at lower enzyme-to-substrate ratios. The inhibition of cellulases by the hydrolysis product, cellobiose, was found to be strongly dependent on the nature of the substrate. The hydrolysis of a low molecular weight model substrate, such as para-nitrophenyl cellobioside, by cellobiohydrolase I is strongly inhibited by cellobiose with a competitive inhibition constant around 20 μM, whereas the hydrolysis of cellulose is more resistant to inhibition with an apparent inhibition constant around 1.5 mM for cellobiose.

Submitted by Lucienne Costa (lucienneferreira@ufcg.edu.br) on 2017-12-13T17:26:54Z No. of bitstreams: 1 CARLOS ALBERTO BISPO DE SOUSA - TESE (PPGEP) 2014.pdf: 2633408 bytes, checksum: 80087f7d1097f18a52b312e44cfc57af (MD5) / Made available in DSpace on 2017-12-13T17:26:54Z (GMT). No. of bitstreams: 1 CARLOS ALBERTO BISPO DE SOUSA - TESE (PPGEP) 2014.pdf: 2633408 bytes, checksum: 80087f7d1097f18a52b312e44cfc57af (MD5) Previous issue date: 2014-11-28 / Capes / A demanda por fontes de energia renovável tem crescido em todo o mundo. Nesse contexto, o bioetanol obtido a partir da hidrólise de materiais lignocelulósicos tem merecido destaque. Porém, a produção das enzimas celulolíticas usadas nesse processo é de custo elevado, e este é o principal empecilho para a obtenção do etanol celulósico em grande escala. O objetivo deste estudo foi produzir enzimas celulolíticas destinadas a produção de bioetanol, a partir da fermentação semissólida da biomassa da palma forrageira pelo fungo filamentoso Trichoderma reesei LCB 48. O estudo da fermentação revelou que a melhor condição foi atingida com umidade inicial de 90% e suplementação de 1% de fonte de nitrogênio. A atividade máxima foi alcançada em 110 horas de processo, com produção de 6,45 U/gds. O estudo de lixiviação das enzimas produzidas revelou como as melhores condições do processo: a relação solvente/substrato de 20g/ml, agitação de 50rpm e tempo de contato de 15 minutos, na qual obteve-se extratos brutos com 15,14 U/gds expressa em carboximetilcelulase (CMCase). As enzimas recuperadas na lixiviação exibiram atividade CMCase ótima em temperatura de 55°C e pH ótimo entre 4,0 e 5,0. Estudos de estabilidade mostraram que a enzima é desativada em valores de pH superiores a 6,5 e temperaturas superiores a 50°C. Ensaios de hidrólise utilisando a própria biomassa da palma e o resíduo da fermentação como material lignocelulósico apresentaram respectivamente produtividade máxima de de 334,4 mg/L.h e 308 mg/L.h de glicose em 4 horas de processo. Foi realizado um estudo de partição das enzimas obtidas utilizando sistemas aquosos bifásicos. O SAB composto por 18% Peg 4000 e 12% citrato de sódio em pH 5,0 resultou em um fator de purificação de 5,31 para a CMCase e 61,4 para celobiase. Para FPase o fator de purificação foi de 2,29 quando a concentração de citrato usada foi de 16%. A biomassa da palma mostrou-se viável tanto para a obtenção das enzimas celulases quanto para a obtenção de açúcares fermentescíveis para produção de bioetanol. Os SABs mostraram-se promissores como etapa inicial de um processo de recuperação e purificação das celulases produzidas. / The demand for renewable energy has grown worldwide. In this context, bioethanol obtained from the hydrolysis of lignocellulosic materials has been highlighted . However, the production of cellulolytic enzymes used in the hydrolysis process is costly, and this is the main obstacle for obtaining cellulosic ethanol on a large scale. This study was designed to produce cellulolytic enzymes production of bioethanol from biomass semisolid fermentation of forage cactus by the filamentous fungus Trichoderma reesei LCB 48. The study revealed that the best fermentation condition was achieved with 90 % humidity and supplementation 1% of the nitrogen source . The maximum activity was achieved in 110 hours of process, with production of 6.45 U/gds . The study of leaching of enzymes produced revealed as the best process conditions: solvent substrate ratio of 20mL/ g , 50 rpm of agitation and contact time of 15 minutes , which was obtained in crude extracts with 15.14 U/gds expressed in carboxymethylcellulase ( CMCase ) . The crude extract was stable for up to 20 days when stored at room temperature. The enzymes recovered in the leaching exhibited CMCase activity at optimal temperature of 55 ° C and optimum pH between 4.0 and 5.0 . Stability studies showed that the enzyme is deactivated at pH values above 6.5 and temperatures above 50 ° C. Hydrolysis assays of pear cactus biomass itself and the residue fermenting lignocellulosic material presented as maximum yield of 334.4 mg/Lh and 308 mg/Lh of glucose in 4 process hours respectively. A study ds enzymes obtained using aqueous two-phase partition systems was carried out . SAB composed of 18 % PEG 4000 and 12% sodium citrate at pH 5.0 resulted in a purification factor of 5.31 to 61.4 for CMCase and cellobiase. FPase for the purification factor was 2.29 when the concentration of citrate used was 16% . Biomass palm proved to be feasible for both obtaining the cellulase enzymes as to obtain fermentable sugars to bioethanol production. The SABs proved promising as an initial step in a process of recovery and purification of cellulases produced .

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-02-07T10:26:14Z No. of bitstreams: 1 TeseCF.pdf: 2944475 bytes, checksum: 0b8b7d83e1195e91eb572351858f7b93 (MD5) / Approved for entry into archive by Camila Passos (camilapassos@ufscar.br) on 2017-02-08T12:04:12Z (GMT) No. of bitstreams: 1 TeseCF.pdf: 2944475 bytes, checksum: 0b8b7d83e1195e91eb572351858f7b93 (MD5) / Approved for entry into archive by Camila Passos (camilapassos@ufscar.br) on 2017-02-08T12:08:29Z (GMT) No. of bitstreams: 1 TeseCF.pdf: 2944475 bytes, checksum: 0b8b7d83e1195e91eb572351858f7b93 (MD5) / Made available in DSpace on 2017-02-08T12:10:12Z (GMT). No. of bitstreams: 1 TeseCF.pdf: 2944475 bytes, checksum: 0b8b7d83e1195e91eb572351858f7b93 (MD5) Previous issue date: 2016-04-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Currently, one of the major challenges for second generation ethanol is to reduce the cost of cellulolytic enzymes. Thus, the development of bioprocesses for the enzyme production on-site and strategies to increase the final yield of the enzymatic hydrolysis are required to ensure that biomass conversion to be economically feasible. Therefore, the objective of this work was to study the production and characterization of enzyme cocktails involved in the degradation of plant biomass by filamentous fungi Trichoderma reesei and Aspergillus niger grown in sequential fermentation and evaluate the application of these cocktails in the saccharification process of sugarcane bagasse. Firstly, evaluation and validation of sequential fermentation cultivation methodology to different strains of Trichoderma. Cultivation were made using sugarcane bagasse "in natura" and pretreated by steam explosion, as a carbon source. The result more significantly was observed for T. reesei Rut C30, the endoglucanase production was 4.2-fold higher than the values obtained in conventional submerged fermentation. The enzyme extracts were characterized in terms of optimum pH and temperature and endoglucanase profile. The thermostability was directly influenced by the type of carbon source and type of cultivation method. Subsequently, the proteomic analysis were performed of enzyme cocktails from T. reesei Rut C30 and A. niger A12 produced by submerged and sequential fermentation in the presence of pretreated bagasse. The performance of the enzyme cocktail in saccharification of pretreated bagasse showed that the combination of enzyme cocktails from T. reesei and A. niger produced by sequential fermentation had a yield than 3-fold higher than the enzyme cocktails of submerged fermentation. In order to evaluate the action of the enzyme cocktails produced by T. reesei and A. niger in sugarcane bagasse saccharification, the last step of the work was to study the additives effects during the sugarcane bagasse hydrolysis aiming at reducing non-productive adsorption of enzymes into lignin. The saccharification results in the presence of soybean protein were 2-fold higher than the controls (no additive) to the enzyme cocktails of two fungi studied produced by solid state fermentation, indicating the potential use of soybean protein as an additive to minimize non-productive adsorption of the enzyme into lignin. Overall, this study presents an interesting final contribution in the cellulase production process and the application of the enzyme cocktail in the hydrolysis of sugarcane bagasse. / Atualmente, um dos grandes desafios para a produção de etanol de segunda geração consiste em diminuir o custo das enzimas celulolíticas. Assim, o desenvolvimento de bioprocessos para produção das enzimas on-site e estratégias para aumentar o rendimento final da hidrólise enzimática são necessários para assegurar que a conversão de biomassa seja economicamente viável. Para tanto, o objetivo deste trabalho foi estudar a produção e caracterização de coquetéis enzimáticos envolvidos na degradação da biomassa vegetal pelos fungos filamentosos Trichoderma reesei e Aspegillus niger cultivados por fermentação sequencial, bem como avaliar a aplicação dos mesmos no processo de sacarificação do bagaço de cana-de-açúcar. Primeiramente foi realizada a avaliação e validação da metodologia de cultivo de fermentação sequencial para diferentes linhagens de Trichoderma. Os cultivos foram feitos utilizando o bagaço de cana “in natura” e prétratado por explosão a vapor, como fonte de carbono. O melhor resultado foi observado para T. reesei Rut C30, em que a produção de endoglucanase foi 4,2 vezes maior do que os valores obtidos em cultivo convencional de fermentação submersa. Os extratos enzimáticos foram caracterizados em termos de pH e temperatura ótimos e perfil de endoglucanase. A termo-estabilidade foi diretamente influenciada pelo tipo de fonte de carbono e tipo de cultivo. Posteriormente, foram realizadas as análises proteômicas dos coquetéis enzimáticos do T. reesei Rut C30 e A. niger A12 produzidos por fermentação submersa convencional e fermentação sequencial, na presença de bagaço de cana prétratado. A performance dos coquetéis enzimáticos na sacarificação do bagaço de cana pré-tratado mostraram que a combinação dos coquetéis enzimáticos de T. reesei e A. niger produzidos por fermentação sequencial tiveram um rendimento 3 vezes maior do que os coquetéis da fermentação submersa. A fim de explorar melhor a ação dos coquetéis enzimáticos produzidos por T. reesei e A. niger na sacarificação do bagaço, na última etapa do trabalho foi estudo o efeito de aditivos durante a hidrólise do bagaco de cana visando à redução da adsorção improdutiva de enzimas na lignina. Os resultados de sacarificação na presença da proteína de soja foram 2 vezes maiores do que os controles (sem aditivo) para os coquetéis enzimáticos dos dois fungos estudados produzidos por fermentação em estado sólido, indicando o potencial do uso da proteína de soja como aditivo para minimizar a adsorção improdutiva das enzimas na lignina. De modo geral, o presente trabalho conseguiu uma contribuição final interessante no processo de produção das celulases e a aplicação do coquetel enzimático na hidrólise do bagaço de cana.