From a metagenomic source to a high-resolution structure of a novel alkaline esterase

Autor(es)

Pereira, Mariana Rangel ; Maester, Thas Carvalho ; Mercaldi, Gustavo Fernando ; de Macedo Lemos, Eliana Gertrudes ; Hyvonen, Marko ; Balan, Andrea

• Tipo de documento: Artigo Científico
• Fonte: Applied Microbiology and Biotechnology; v. 101, n. 12, p. 4935-4949, JUN 2017.
• Citações Web of Science: 10

Resumo

Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of alpha/beta hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-C resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met(213) and Phe(217)) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues. (AU)

• Processo FAPESP: 11/09136-7 - Caracterização Funcional e Estrutural de Enzimas Lipolíticas de um Consórcio Microbiano Degradador de Óleo Diesel
• Beneficiário: Mariana Rangel Pereira
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 12/20490-0 - Caracterização do mecanismo de interação com substratos de lipases/esterases termoestáveis e desenho racional para desenvolvimento de proteínas com alto potencial biotecnológico
• Beneficiário: Mariana Rangel Pereira
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado