Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/332148
Type: TESE DIGITAL
Degree Level: Doutorado
Title: Investigação de vias de degradação de biomassa lignocelulósica no cupim Coptotermes gestroi complementares a hidrolases glicosídicas visando aplicação na produção de bioprodutos : Investigation of lignocellulosic degradation by the termite Coptotermes gestroi complementary to glycoside hydrolases aiming at the production of bioproducts
Title Alternative: Investigation of lignocellulosic degradation by the termite Coptotermes gestroi complementary to glycoside hydrolases aiming at the production of bioproducts
Author: Franco Cairo, João Paulo Lourenço, 1984-
Advisor: Squina, Fabio Marcio
Abstract: Resumo: Cupins sao insetos sociais que vivem em colonias dividindo o trabalho entre suas castas e tem a sua dieta baseada no consumo de material lignocelulosico. Os cupins sao considerados os menores biorreatores da Terra, e um dos organismos mais eficiente na decomposicao de lignoceluloses. A degradacao da biomassa vegetal pode atingir cerca de 75- 90% de eficiencia e a lignina e modificada. A digestao ocorre no trato digestivo, no qual ocorre a secrecao de enzimas ativas em carboidratos (CAZymes), entre elas, as hidrolases glicosidicas tais como celulases e hemicelulases. Essas enzimas sao secretadas no intestino anterior e medio, e no posterior pelos simbiontes que ali habitam, tais como protozoarios e bacterias. O objetivo dessa tese foi investigar a ocorrencia de mecanismos complementares a hidrolases glicosidicas no digestoma do cupim Coptotermes gestroi, principalmente mecanismos oxido-redutores, os quais possam explicar a eficiencia na degradacao de lignoceluloses. Neste trabalho de doutorado, primeiramente duas hidrolases glicosidicas endogenas de C. gestroi, uma Endo-¿À-1,4-glicosidase-CgEG-1 (GHF-9) e uma ¿À-1,4- glicosidase-CgBG-1 (GHF-1) foram funcional e estruturalmente caracterizadas. Os resultados mostraram que as duas enzimas atuam em conjunto no digestoma de C. gestroi e possuem alto grau de sinergia na degradacao de polissacarideos naturais e lignocelulose complexa, entretanto, a sacarificacao e muito baixa em comparacao com celulases fungicas. Assim, com o objetivo de ampliar o conhecimento do arsenal de enzimas CAZy de C. gestroi, analises de metatranscriptomica e metaproteomica foram realizadas nas castas operaria e de soldados desse inseto. Os dados revelaram que ambas as castas possuem um arsenal de enzimas CAZy similar, incluindo enzimas de Atividade Auxiliares-AAs (lacases e glicose oxidases), bem como oxido-redutases relacionadas a processos de Pro-oxidacao, Antioxidacao e Detoxificacao (PAD). Alem disso, a maioria dos gene/enzimas CAZy e PAD tiveram maior abundancia na casta operaria, sugerindo um papel na digestao da lignocelulose. Posteriormente, uma analise do transcriptoma de operarios C. gestroi submetidos a dieta com diferentes bagacos de cana-de-acucar, revelou que a expressao de genes que codificam superoxido dismutases (genes PAD), foi modulado pela dieta. Desta forma, o gene endogeno CGSOD-1 foi clonado e sua proteina CgSOD-1 foi caracterizada funcional e biologicamente. CgSOD-1 e uma dismutase classica, que apresenta sinergia com CgEG-1 na hidrolise de ¿À- glucano. CgSOD-1 foi imunolocalizada nos segmentos anteriores e medio do trato digestivo de C. gestroi, mesma localizacao de CgEG-1, sugerindo assim o papel dessa SOD no digestoma de C. gestroi. Analises bioquimicas revelaram ainda que CgSOD-1 atua na clivagem de polissacarideos, atraves da geracao de especies reativas de oxigenio. Mais que isso, CgSOD-1 foi utilizada para suplementar coqueteis celuloliticos, revelando um aumento de 30% na sacarificacao de biomassa. De forma geral, esta tese mostrou que as hidrolases glicosidicas produzidas pelo cupim inferior C. gestroi nao sao eficientes na degradacao de lignocelulose complexa, e que existem enzimas complementares ou auxiliares as hidrolases glicosidicas, principalmente oxido-redutases, que atuam na conversao da biomassa lignocelulosica no digestoma desses insetos

Abstract: Termites are social insects living in colonies and dividing the labor among the castes. This insect has its diet based on the consumption of lignocelluloses. The worker caste is responsible for feeding the colony, which turn the termites into on of the smaller bioreactor on Earth, and one of the most efficient organisms in the decomposition of lignocelluloses. The degradation of this material achieve approximately 75-90% efficiency and the lignin is modified. The digestion occurs in the termite gut, which occurs the secretion of carbohydrateactive enzymes (CAZymes), such as glycoside hydrolases (cellulases and hemicellulases). These enzymes are secreted in the fore- and midgut, and also by the symbionts, such as protozoa and bacteria, in the hindgut. However, these cellulases have been described in the literature as inefficient into degrading recalcitrant lignocellulose. Thus, the aim of this thesis was to investigate the occurrence of complementary mechanisms to glycoside hydrolases in the digestome the termite Coptotermes gestroi, mainly redox mechanisms, which could explain the efficient lignocelluloses degradation by termites. Thus, two endogenous glycoside hydrolases from C. gestroi, an endo-1,4-â-glucosidase CgEG-1 (GHF-9) and a â-1,4- glucosidase CgBG-1 (GHF-1) were functionally and structurally characterized. The results showed that the two enzymes act in synergy in the C. gestroi¿s digestome, exhibiting a high degree of synergy in the degradation of polysaccharides and complex lignocellulose. However, the level of biomass saccharification was very low, in comparison to saccharification yields using fungal cellulases. Thus, aiming to explore the potential of CAZymes in C. gestroi, analysis of metatranscriptomic and metaproteomics were performed in the worker and soldier castes. The results revealed that both castes had a similar repertoire of CAZymes, including from the Auxiliaries Activities (AAs) group (laccases, and glucose oxidases) and oxidoreductases related to Pro-oxidation, Antioxidant, and Detoxification (PAD) processes. In addition, genes and enzymes classified as CAZy and PAD had higher abundance in the worker caste, suggesting a role of theses enzymes in lignocellulose breakdown. Subsequently, a transcriptome analysis of C. gestroi¿s workers, fed on different sugarcane bagasses, have revealed that the expression of genes encoding superoxide dismutases, a PAD enzyme, was modulated by the diet. Thus, the endogenous CGSOD-1 gene was cloned and the protein CgSOD-1 was functional and biologically characterized. CgSOD-1 is a typical SOD, showing synergy with CgEG-1 in the hydrolysis of â-glucan. CgSOD-1 was immunolocalized in the fore- and midgut of C. gestroi, as well as CgEG-1, thus, suggesting the role of this SOD in the digestome. Biochemical analyzes also revealed that CgSOD-1 acts on the cleavage of polysaccharides, throughout the generation of reactive oxygen species. Moreover, CgSOD-1 was used to supplement cellulolytic cocktails, revealing a 30% increase in the biomass saccharification. In conclusion, this thesis has shown that the glycoside hydrolases produced by the lower termite C. gestroi are not efficient in the degradation of complex lignocellulose, moreover, it was evidenced that C. gestroi synthesized auxiliary enzymes to glycoside hydrolases, primarily oxidoreductases, which could act in the conversion of lignocellulosic biomass in the digestome of this insect
Subject: Produtos biológicos
Térmita
Biomassa vegetal
Glicosídeo hidrolases
Enzimas
Language: Multilíngua
Editor: [s.n.]
Citation: FRANCO CAIRO, João Paulo Lourenço. Investigação de vias de degradação de biomassa lignocelulósica no cupim Coptotermes gestroi complementares a hidrolases glicosídicas visando aplicação na produção de bioprodutos: Investigation of lignocellulosic degradation by the termite Coptotermes gestroi complementary to glycoside hydrolases aiming at the production of bioproducts. 2016. 1 recurso online ( 171 p.). Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia, Campinas, SP.
Date Issue: 2016
Appears in Collections:IB - Tese e Dissertação

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