Bioquímica e Biologia Molecular
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11837
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Resultados da Pesquisa
Item Extracellular α-Galactosidase from Debaryomyces hansenii UFV-1 and Its use in the hydrolysis of raffinose oligosaccharides(Journal of Agricultural and Food Chemistry, 2006-02-17) Rezende, Sebastião T. de; Marques, Virgínia M.; Trevizano, Larissa M.; Passos, Flávia M. L.; Oliveira, Maria G. A.; Bemquerer, Marcelo P.; Oliveira, Jamil S.; Guimarães, Valéria M.; Viana, Pollyanna A.Raffinose oligosaccharides (RO) are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. ROs are hydrolyzed by α-galactosidases that cleave α-1,6-linkages of α-galactoside residues. The objectives of this study were the purification and characterization of extracellular α-galactosidase from Debaryomyces hansenii UFV-1. The enzyme purified by gel filtration and anion exchange chromatographies presented an Mr value of 60 kDa and the N-terminal amino acid sequence YENGLNLVPQMGWN. The Km values for hydrolysis of pNPαGal, melibiose, stachyose, and raffinose were 0.30, 2.01, 9.66, and 16 mM, respectively. The α-galactosidase presented absolute specificity for galactose in the α-position, hydrolyzing pNPGal, stachyose, raffinose, melibiose, and polymers. The enzyme was noncompetitively inhibited by galactose (Ki = 2.7 mM) and melibiose (Ki = 1.2 mM). Enzyme treatments of soy milk for 4 h at 60 °C reduced the amounts of stachyose and raffinose by 100%.Item Debaryomyces hansenii UFV-1 intracellular α-Galactosidase characterization and comparative studies with the extracellular enzyme(Journal of Agricultural and Food Chemistry, 2009-02-18) Rezende, Sebastião T. de; Viana, Pollyanna A.; Passos, Flávia Maria Lopes; Oliveira, Jamil S.; Teixeira, Kádima N.; Santos, Alexandre M. C.; Bemquerer, Marcelo P.; Rosa, José C.; Santoro, Marcelo M.; Guimarães, Valéria M.Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular α-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS−PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the α-galactosidases were identical. Intracellular α-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular α-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s^−1, respectively) for the p-nitrophenyl-α-d-galactopyranoside substrate. The Km for hydrolysis of pNPαGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was acompetitively inhibited by galactose (Ki = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 °C reduced stachyose and raffinose amounts by 100 and 73%, respectively.Item Activity of Debaryomyces hansenii UFV-1 a-galactosidases against a-D-galactopyranoside derivatives(Carbohydrate Research, 2011-04-01) Rezende, Sebastião T. de; Viana, Pollyanna A.; Alves, Arianne de A.; Manfrini, Rozângela M.; Alves, Ricardo J.; Bemquerer, Marcelo P.; Santoro, Marcelo M.; Guimarães, Valéria M.α-d-Galactopyranosides were synthesized and their inhibitory activities toward the Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases were evaluated. Methyl α-d-galactopyranoside was the most potent inhibitor compared to the others tested, with values of 0.82 and 1.12 mmol L−1, for extracellular and intracellular enzymes, respectively. These results indicate that the presence of a hydroxyl group in the C-6 position of α-d-galactopyranoside derivatives is important for the recognition by D. hansenii UFV-1 α-galactosidases.Item Hydrolysis of soybean isoflavones by Debaryomyces hansenii UFV-1 immobilised cells and free β-glucosidase(Food Chemistry, 2014-03-01) Maitan-Alfenas, Gabriela P.; Lage, Lorena G. de A.; Almeida, Maíra N. de; Visser, Evan M.; Rezende, Sebastião T. de; Guimarães, Valéria M.An intracellular β-glucosidase from Debaryomyces hansenii UFV-1 was produced in an YP medium with cellobiose as the carbon source. This enzyme was purified, characterised and presented a Mr of 65.15 kDa. Yeast cells containing the intracellular β-glucosidase were immobilised in calcium alginate. The free β-glucosidase and immobilised cells containing the enzyme presented optima values of pH and temperature of 6.0 and 45 °C and 5.5 and 50 °C, respectively. The free enzyme maintained 62% and 47% of its original activity after 90 days at 4 °C and after 15 days at room temperature, respectively. The immobilisation process resulted in higher enzyme thermostability at 45 and 50 °C. Soy molasses treatment with the free enzyme and the immobilised cells containing β-glucosidase, for 2 h at 40 °C, promoted efficient hydrolysis of isoflavone glicosides to their aglycon forms. The results suggest that this enzyme could be used in the food industry, in the free or immobilised forms, for a safe and efficient process to hydrolyse isoflavone glycosides in soy molasses.