ABSTRACT
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 K(i)(') values of 0.82 and 1.12 mmolL(-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.
Subject(s)
Debaryomyces/enzymology , Galactose/metabolism , Galactosidases/metabolism , Galactose/analogs & derivatives , Molecular StructureABSTRACT
Spectroscopic and thermodynamic properties were determined for Debaryomyces hansenii UFV-1 extracellular and intracellular alpha-galactosidases. alpha-Galactosidases showed similar secondary structure compositions (alpha-helix, beta-sheet parallel and beta-turn). Effects of pH and temperature on the structure of alpha-galactosidases were investigated using circular dichroism spectroscopy. It was more pronounced at low pH. Microcalorimetry was employed for the determination of thermodynamic parameters. Immediate thermal denaturation reversibility was not observed for alpha-galactosidases; it occurred as a thermodynamically driven process. Extracellular alpha-galactosidase, at pH 5.5, showed lower T(m) when compared to the intracellular enzyme. The CD and DSC data suggest that D. hansenii alpha-galactosidases have different behaviors although they possess some similar secondary structures.
Subject(s)
Circular Dichroism , Debaryomyces/enzymology , alpha-Galactosidase/chemistry , Calorimetry, Differential Scanning , Extracellular Space/enzymology , Hydrogen-Ion Concentration , Protein Denaturation , Protein Structure, Secondary , Temperature , Thermodynamics , Transition Temperature , alpha-Galactosidase/metabolismABSTRACT
Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-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 alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher kcat than the intracellular enzyme (7.16 vs 3.29 s-1, respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The Km for hydrolysis of pNPalphaGal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was a competitively 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 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.
Subject(s)
Debaryomyces/enzymology , alpha-Galactosidase/chemistry , alpha-Galactosidase/metabolism , Amino Acid Sequence , Carbohydrates/analysis , Enzyme Stability , Intracellular Space/enzymology , Kinetics , Molecular Sequence Data , Molecular Weight , Oligosaccharides/metabolism , Soy Milk/chemistry , Substrate SpecificityABSTRACT
Raffinose oligosaccharides (RO) are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. ROs are hydrolyzed by alpha-galactosidases that cleave alpha-1,6-linkages of alpha-galactoside residues. The objectives of this study were the purification and characterization of extracellular alpha-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 pNP alphaGal, melibiose, stachyose, and raffinose were 0.30, 2.01, 9.66, and 16 mM, respectively. The alpha-galactosidase presented absolute specificity for galactose in the alpha-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 degrees C reduced the amounts of stachyose and raffinose by 100%.
