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Master's Dissertation
DOI
10.11606/D.97.2018.tde-03122018-183322
Document
Author
Full name
Bruno Alves França
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Lorena, 2018
Supervisor
Committee
Segato, Fernando (President)
Damasio, André Ricardo de Lima
Ferraz, André Luis
Kadowaki, Marco Antonio Seiki
Title in Portuguese
Produção de LPMOs recombinantes do fungo Thermothelomyces thermophila M77 e seu efeito na sacarificação enzimática do bagaço de cana
Keywords in Portuguese
Thermothelomyces thermophila
Biomassa lignocelulósica
Expressão heteróloga
LPMOs
Oxidação da holocelulose
Abstract in Portuguese
A biomassa lignocelulósica é uma fonte abundante de açúcares simples passíveis de serem fermentados em uma variedade de bioprodutos de maior valor agregado, além do etanol de segunda geração. Tal diversidade é relevante ao desenvolvimento e aprimoramento do conceito de biorrefinarias e da bioeconomia, em um viés mais amplo. Todavia, a elevada recalcitrância dos lignocelulósicos dificulta a sua sacarificação enzimática, resultando em bioprocessos mais onerosos. Por isso, coquetéis com diferentes enzimas ativas em carboidrato (CAZymes) são desenvolvidos, em busca de uma maior eficiência e melhor relação custo/benefício, para processos em larga escala. Dentre as CAZymes estudadas, encontram-se as mono-oxigenases líticas de polissacarídeo (LPMOs), tendo em vista a sua atestada capacidade de otimizar a hidrólise da lignocelulose, quando em sinergismo com diversas hidrolases. Levando isto em conta, selecionou-se, ao atual estudo, o ascomiceto termofílico Thermothelomyces thermophila (anteriormente denominado Myceliophthora thermophila), pois este tem se mostrado capaz de expressar e secretar ampla gama de LPMOs ativas em diferentes substratos. Objetivando-se estudar duas LPMOs derivadas deste organismo, as mesmas foram expressas, heterologamente, por Aspergillus nidulans linhagem A773, utilizando-se o vetor de expressão pEXPYR construído para viabilizar a secreção de altas concentrações de proteínas recombinantes. As proteínas heterólogas aqui analisadas foram denominadas TtLPMO1A9 e TtLPMO2A9. Embora ambas tenham sido capazes de gerar peróxido de hidrogênio na presença de oxigênio molecular e de um doador de elétrons, apenas TtLPMO2A9 apresentou atividade contra substratos celulósicos e bagaço de cana pré-tratado hidrotermicamente, atuando, em associação com hidrolases homemade e o preparo enzimático comercial Celluclast 1.5L, a degradação de tais materiais.
Title in English
Production of recombinant LPMOs from the fungus Thermothelomyces thermophila M77 and their effect over the enzymatic saccharification of sugar cane bagasse
Keywords in English
Thermothelomyces thermophila
Heterologous expression.
Holocellulose oxidation
Lignocellulosic biomass
LPMOs
Abstract in English
The lignocellulosic biomass is an abundant source of simple sugars that can be fermented to various value-added bio-based products. This diversity is seen as relevant to the improvement of biorefinery and bioeconomy concept. Nevertheless, the significant recalcitrance of lignocellulose imposes dificulties to its enzymatic saccharification, resulting in onerous bioprocessing. This scenario stimulates studies based on the development of efficient and cost-effective customizable carbohydrate-active enzyme (CAZymes) cocktails for large-scale processes. Among the available CAZymes, there are the lytic polysaccharide monooxygenases (LPMOs), a set of oxidative proteins capable of optimizing the lignocellulose hydrolysis, when acting in synergism with various hydrolases. Based on this fact, in the current study, the thermophilic ascomycete Thermothelomyces thermophila (previously known as Myceliophthora thermophila) was adopted, because of its ability of expressing and secreting large amounts of LPMOs. Thus, two LPMOs derived from this fungus was heterologously produced by an expression system composed by Aspergillus nidulans strain A773 and the vector pEXPYR: an expression vector built to increase the secretion of recombinant proteins. The heterologous proteins herein analysed were termed as TtLPMO1A9 and TtLPMO2A9. Although both enzymes were able to produce hydrogen peroxide in the presence of molecular oxygen and an electron donor, only the second one was active in reactions with cellulosic substrates and hydrothermally pre-treated sugar cane bagasse. When tailor-made hydrolases and the commercial enzymatic mixture Celluclast 1.5L were supplemented with TtLPMO2A9, it was noticed na improvement of the deconstruction of the aforementioned substrates.
 
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Publishing Date
2018-12-03
 
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