Caracterização bioquímica e estudos estrutural e termodinâmico de α-galactosidases de Debaryomyces hansenii UFV-1

• Autor(a) principal: Viana, Pollyanna Amaral
• Data de Publicação: 2009
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/325
• topic: Alfa-galactosidases; Debaryomyces hansenii; α-galactosidases; Debaryomyces hansenii; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

Human consumption of soy-derived products has been limited by the presence of nondigestible oligosaccharide, such as the α-galacto-oligosaccharides raffinose and stachyose (GO). Most mammals, including man, lack pancreatic α-galactosidase, which is necessary for the hydrolysis of these sugars. However, GO can be fermented by gas-producing microorganisms present in the large intestine, which in turn can induce flatulence and other gastrointestinal disorders. The use of microorganisms expressing α-galactosidase is promising solution to the elimination of GO before they reach the large intestine. In the present study, we reported the production and purification of Debaryomyces hansenii UFV-1 extracellular and intracellular αα-galactosidases, for studies of molecular (circular dichroism) and kineticbiochemical characterizations of these enzymes. We also performed the thermodynamic analysis using microcalorimetry (DSC), aiming for better knowing the individual characteristics of each α-galactosidase, with the purpose for using these enzymes in the elimination of the GO in soy-derived products. Debaryomyces hansenii UFV-1 cells, cultivated on galactose as carbon source, produced extracellular and intracellular α- galactosidases, with 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDSPAGE), and 5.15 and 4.15 pI values, respectively. Extracellular and intracellular α- galactosidases deglycosylated presented 36 and 40 kDa molecular mass, with 40 and 34 % carbohydrate content, respectively. The N-terminal sequences of the α-galactosidases were identical. Amino acid analysis from D. hansenii UFV-1 intracellular and extracellular α- galactosidases showed few variations in their contents. 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 pNPαGal substrate. The intracellular enzyme Kcat values for melibiose, stachyose, and raffinose were 0.03, 6.05 and 4.12 s-1, respectively. Incubation of intracellular isoform with soymilk for 6 h at 55 oC reduced stachyose and raffinose amounts by 100 and 73 %, respectively. It can be observed that D. hansenii UFV-1 α-galactosidase was efficient in reducing the GO presents in soy-derived products, and it is appropriate for industrial use in the processing of these sugars. α-D-Galactopyranosides derivatives were synthesized for elucidate inhibitory activities of D. hansenii UFV-1 α-galactosidases. Methyl α-D-galactopyranoside was the most potent inhibitor compared to the others tested, with Ki values of 0.82 and 1.12 mM, for extracellular and intracellular enzymes, respectively. The presence of hydroxyl group in the C-6 position was important for the recognition by D. hansenii UFV-1 α-galactosidases. The glycoproteins were completely hydrolyzed with 6 M HCl at 80 °C, releasing the monosaccharides after 2 h of incubation. The presence of galactose and mannose was observed in the extracellular α-galactosidase and xylose in the intracellular enzyme. Spectroscopic, kinetic and thermodynamic properties were determined for D. hansenii UFV-1 α-galactosidases. Effects of pH and temperature on the structure of these enzymes were investigated using Circular Dichroism (CD). D. hansenii UFV-1 α- galactosidases showed very similar secondary structure compositions (α-helix, β-sheet paralel and β-turn). Differential Scanning Calorimetry (DSC) was employed for the determination of some thermodynamic parameters during protein denaturation. Thermal denaturation reversibility was not immediately observed for D. hansenii UFV-1 α-galactosidases; however it occurred as a thermodynamically controlled process. Extracellular α-galactosidase, at pH 5.5, showed a Tm value smaller (86.1 °C) when compared to the intracellular enzyme (87.3 °C). The cooperativity relative index for intracellular α-galactosidase (ΔT1/2 = 5.0 ºC) was smaller than that of the extracellular enzyme (ΔT1/2 = 5.6 ºC). Thus, the CD and DSC data suggest that the two enzymes have different and discrete behaviors although they possess similar secondary structure. The related Arrhenius activation energy for extracellular and intracellular α-galactosidase from D. hansenii UFV-1 (88 and 95 KJ/mol, respectively) was correspondingly high and indicates a considerable transient chemical change during the binding process. The conditions tested for D. hansenii UFV-1 extracellular α-galactosidase crystallization were the Kits 1 and 2 (Crystal Screen) using the vapor diffusion technique in hanging drop. It had crystal appearance in determined conditions; however, the X-ray diffraction analysis had indicated salts formation used in the crystallization.