Prospecção e caracterização de enzimas envolvidas na desconstrução de biomassa a partir de abordagem metagenômica

• Ano de defesa: 2023
• Autor(a) principal: Sales, Rodrigo Mauricio Marinsek
• Orientador(a): Quirino, Betania Ferraz
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Católica de Brasília
• Programa de Pós-Graduação: Programa Stricto Sensu em Ciências Genômicas e Biotecnologia
• Departamento: Escola de Saúde e Medicina
• País: Brasil
• Palavras-chave em Português: Metagenoma; Ligninases; β-glicosidases; Biomassa
• Palavras-chave em Inglês: Metagenome; Ligninases; Biomass
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS
• Link de acesso: https://bdtd.ucb.br:8443/jspui/handle/tede/3338

Lignocellulosic biomass is composed of three main polymers, which are cellulose, hemicellulose and lignin, with cellulose being the most abundant organic compound in nature and lignin being the most abundant phenolic compound in nature. The depolymerization of this renewable raw material is the first step towards obtaining chemical compounds of industrial interest and one of the most promising technologies for this deconstruction involves the use of enzymes. The identification of new enzymes with high potential for the deconstruction of biomass favors the development of environmentally benign processes. With metagenomic approaches and molecular biology techniques, it is possible to explore the microbial diversity (culturable and non-culturable) of different environments and thus find enzymes with biotechnological potential for biomass degradation. The objective of this work was to express, purify and biochemically characterize a β-glucosidase with biotechnological potential for the industrial market and to carry out amplification and cloning in an expression vector of at least one ligninase. β-glucosidase (Bgl08) was produced from a goat rumen metagenomic library constructed in previous studies by the Embrapa Agroenergia research team and presented the following characteristics: 1) activity against the substrates pNPG, pNPX, cellobiose and salicin; 2) optimum temperature of 45 °C; 3) optimum pH of 6.0; 4) Vmax(pNPG) of 3.10-6 mmol/min (U) and Km of 1.83 mM ± 0.26; 5) Vmax (cellobiose) 7.05.10- 3 mmol/min (U) and Km of 7.49 mM ± 1.59. The search for ligninase was carried out using microbial consortia using the lignin enrichment technique. With the total DNA extracted from this consortium, a metagenomic library was constructed and three pairs of primers corresponding to Dyp enzymes and five pairs of primers corresponding to SOD enzymes were designed and synthesized so that these genes could be amplified and cloned into expression vectors.