Produção, purificação e caracterização de β-glicosidases livre e imobilizada de Debaryomyces hansenii UFV-1

• Ano de defesa: 2011
• Autor(a) principal: Maitan, Gabriela Piccolo
• Orientador(a): Guimarães, Valéria Monteze
• Banca de defesa: Oliveira, Eduardo Basílio de , Fietto, Luciano Gomes
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: β-glicosidases livre; Debaryomyces hansenii
• Palavras-chave em Inglês: β-glucosidases; Debaryomyces hansenii
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
• Link de acesso: http://locus.ufv.br/handle/123456789/2429

The objectives of this work were to establish the best conditions for Debaryomyces hansenii UFV-1 yeast cultivation for β-glucosidase production, purification and characterization. In addition, another objective was to immobilize the yeast cells containing the intracellular β-glucosidase in calcium alginate and characterize the immobilized enzyme. The yeast produced an intracellular β-glucosidase when grown for 12 h in YP medium containing cellobiose as carbon source. The enzyme was purified by ion exchange, gel filtration and hydrophobic interaction chromatografies and at the end of the process, the β-glucosidase showed a purification factor of 99.3 times, with a yield of 8 %. The molecular mass was approximately 198 kDa when estimated by SDS-PAGE, and approximately 178 kDa when estimated by gel filtration. The maximum activity occurred at pH 6.0 and temperature of 45 °C. The intracellular β-glucosidase half-life values were 356, 312, 73 and 5 minutes, at temperatures of 40, 45, 50 and 55 °C, respectively. The KM and Vmax values using the substrate ρNPβGlc were 0.434 mM and 0.021 mM/min, respectively. The enzyme showed absolute specificity for glucose in β position and the enzymatic activity was completely inhibited by ferric chloride, silver nitrate and SDS, all of them at final concentrations of 0.2 and 2 mM. The yeast cells were immobilized in calcium alginate and the enzyme showed pH and temperature optima of 5.5 and 50 °C, respectively. The immobilization provided higher enzyme thermostability at temperatures of 45 and 50 °C. The KM app and Vmax app. values, with the substrate ρNPβGlc, were 4.35 mM and 3.63 mmol of ρNP/min/g cell, respectively. D. hansenii is a nonpathogenic yeast and it is found in various types of food. So, the use of immobilized cells of this yeast in calcium alginate in food industry would not cause problems for human health. In this case, the enzyme is more stable and the process is more economical because there is no spending on enzyme purification and also the possibility of reuse of alginate beads containing the enzyme β-glucosidase.