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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2020.tde-11022020-141425
Document
Author
Full name
Paula Zaghetto de Almeida
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2019
Supervisor
Committee
Polizeli, Maria de Lourdes Teixeira de Moraes (President)
Oliva Neto, Pedro de
Segato, Fernando
Ward, Richard John
Title in Portuguese
Clonagem e expressão heteróloga de enzimas do sistema celulolítico visando a degradação de biomassa lignocelulósica
Keywords in Portuguese
Anoxybacillus thermarum
b-glucosidase
Celulases
Endoglucanase
Eubacterium cellulosolvens
Hidrólise
Trichoderma reesei
Abstract in Portuguese
O Brasil possui um grande potencial para a produção de biocombustíveis a partir de biomassa lignocelulósica. Neste contexto para o desenvolvimento de um bioprocesso limpo e sustentável faz-se necessária a utilização de grandes quantidades de enzimas, sobretudo as celulases. As celulases são responsáveis pela quebra da cadeia de celulose em unidades de glucose. Para baratear e ampliar a produção destas enzimas uma das estratégias que podem ser adotadas é a expressão heteróloga. Neste trabalho foram expressas em Escherichia coli uma endoglucanase de Trichoderma reesei (TR), uma endoglucanase de Eubacterium cellulosolvens com a estrutura completa (MI+MII) e parcial (MII) e uma ?-glucosidase de Anoxybacillus thermarum (BG). A enzima TR, que foi expressa com a cepa de E. coli Origami2, destacou-se por ter pH ótimo baixo, de 3,5 e temperatura ótima de 65°C, ter uma excelente estabilidade entre os pH 2,5 e 7, e ser ativada em 88% por Mn2+. As endoglucanases MI+MII e MII se destacaram pela produção fácil e rápida, gerando grande quantidade de enzimas com baixo custo, possuem pH ótimo de 4,5 e temperatura de 45°C para MI+MII e 50°C para MII. Ambas em extrato bruto são estáveis nos pH de 4 a 7 e temperaturas de 40°C e 50°C por até 24 horas. A BG destacouse, além da produção rápida e pouco dispendiosa, pela excelente tolerância ao produto, sendo a mesma ativada em concentrações de até 0,4 M de glucose. Seu pH ótimo foi de 7 e a temperatura de 65°C, o km de 0,35 mM de ?NPG e Vmáx cerca de 7000 U/mg, variando pouco conforme as diferentes metodologias aplicadas. A BG foi estável entre os pH 5,5 a 8 e na temperatura de 50°C por até 48 horas. As enzimas TR e BG foram aplicadas na degradação de bagaço-de-cana e capim-elefante, in natura ou tratados por autohidrólise, juntamente com um extrato bruto de enzimas provenientes do cultivo de Aspergillus brasiliensis. Os melhores rendimentos foram obtidos na hidrólise com o capim-elefante in natura, resultando na produção até 30 µmol/mL de açúcares redutores.
Title in English
Cellulolytic system enzymes cloning and heterologous expression aiming the lignocellulosic biomass degradation
Keywords in English
Anoxybacillus thermarum
b-glucosidase
Cellulases
Endoglucanase
Eubacterium cellulosolvens
Hydrolysis
Trichoderma reesei
Abstract in English
Brazil has great potential for the biofuels production from lignocellulosic biomass. In this context, the development of a clean and sustainable bioprocess requires the use of large quantities of enzymes, especially cellulases. Cellulases are responsible for the breakdown of cellulose chain into glucose units. To increase the production of enzymes and turn the process cheaper one of the strategies that can be adopted is the heterologous expression. In this work, an endoglucanase from Trichoderma reesei (TR), another endoglucanase from Eubacterium cellulosolvens, with complete (MI+MII) and partial (MII) structure, and a ?-glucosidase from Anoxybacillus thermarum (BG) were expressed in Escherichia coli. The enzyme TR, which was expressed with E. coli Origami2, stood out to have a low optimal pH of 3.5 and an optimal temperature of 65°C, to have an excellent pH stability, among 2.5 and 7, and to be activated in 88% by Mn2+. The MI+MII and MII endoglucanases were characterized by easy and fast production, generating a large quantity of enzymes with low cost, having an optimum pH of 4.5, and an optimal temperature of 40°C for MI+ MII, and 50°C for MII. The crude extract of both were stable at pH 4 to 7, and 40ºC and 50°C for up to 24 hours. The BG had a fast and inexpensive production, and an excellent tolerance to the product. BG was activated up to 0.4 M of glucose. Its optimum pH was 7 and the temperature was 65°C. The km was calculated as 0.35 mM ?NPG and Vmax about 7000 U/mg, varying little according to the methodology applied. BG was stable among pH 5.5 to 8 and at 50 ° C for up to 48 hours. The TR and BG were added to a crude extract of enzymes from Aspergillus brasiliensis, and applied in the degradation of sugarcane bagasse and elephantgrass, both in natura or treated by autohydrolysis. The best hydrolysis yields were obtained with elephantgrass in natura, resulting in the production up to 30 µmol/mL of reducing sugars.
 
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Publishing Date
2020-04-28
 
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