Purificação parcial e caracterização bioquímico-cinética de α-galactosidase de Aspergillus terreus

• Autor(a) principal: Ferreira, Joana Gasperazzo
• Data de Publicação: 2007
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2468
• topic: α-galactosidase; Aspergillus terreus; Purificação; α-galactosidase; Aspergillus terreus; Purification; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

The α-galactosidase has the potencial to hydrolyze α-1,6 linkages in galactose oligosaccharides (GO). These oligosaccharides are considered the major factors responsible for flatulence following ingestion of soybean and other seed legumes, due to the absence of this enzyme in the intestinal mucous membrane of men and monogastrics animals. Therefore, the most important factor for improvement of the soy nutritional value is to eliminate the GO from soy products. The aims of this work was to produce, purify and characterize one extracellular isoform of α-galactosidase from Aspergillus terreus and evaluate the hydrolysis of GO presents in soybean milk. The fungus A. terreus was cultivated in mineral medium containing wheat bran as carbon source for 7 days at 28 °C. The enzymatic extracts were submitted to the chromatography in Sephacryl S-200, Phenyl-Sepharose and DEAE-Sephacel resins. The last purification step resulted in a purification factor of 26.96 times with a recovery of 19.07 %. A molecular mass of 50 kDa was determined by SDS-PAGE white the elution of the α-galactosidase from Sephacryl S-200 showed a molecular mass of 77.3 kDa. The maximum activities of the α-galactosidase were detected in pH 5.0 at 55 °C. The enzyme maintained 90 % of its original activity after incubation in pH 4 6 at 40 °C. The α-galactosidase lost 39 % of its initial activity after preincubation for 151 hours at 50 °C. At 55 °C the enzyme maintained 33 % of this original activity after 52 hours and at 60 °C 92 % of its initial activity was maintained for 30 minutes. The half-lives at 55 and 60 °C were 35 hours and 103 minutes, respectively. The relative rate of hydrolysis of various substrates were as follows ρ-NP-αGal > melibiose > m-NP-αGal > raffinose > stachyose > locust bean gum > guar gum. The values of KM, ap for ρ-NP-αGal, melibiose, raffinose and stachyose were 0.75, 7.39, 32.99 and 54.74 mM, respectively. The enzyme was totally inhibited by silver nitrate, and partially inhibited by Cu2+ and galactose. In the presence of the substrate ρ-NP-αGal the enzyme showed a competitive inhibition by galactose (Ki 0,61 mM). The energy of activation for ρ-NP-αGal, melibiose, raffinose, and stachyose were 65.85, 39.77, 42.98 and 47.27 kJ/mol, respectively. The α-galactosidase was not able to convert erythrocytes of group B blood cells to group O type cells. The results of treatments of soy milk at 50 °C with α-galactosidase semi-purified for 12 hours showed reduction of 100 % in the raffinose and 60.3 % in the stachyose. Therefore, it can be observed that the α-galactosidase from A. terreus was efficiently reduced the GO present in soy milk.