Purificação, caracterização bioquímica e aplicações biotecnológicas de α-galactosidases de Aspergillus terreus

• Autor(a) principal: Reis, Angélica Pataro
• Data de Publicação: 2007
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/285
• topic: α-galactosidases; Aspergillus terreus; Caracterização bioquímica; α-galactosidases; Aspergillus terreus; Biochemical characterization; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

The enzyme α-galactosidase (α-D-galactoside galactohydrolase, EC 3.2.1.22) catalyzes the cleavage of α-1,6-galactosyl terminal residues present in different substrates, including linear and branching oligosaccharides, polysaccharides and synthetic substrates such as p-hitrophenyl-α-1,6-galactosyl -D- galactopyranoside (ρNPαGal). The α-galactosidase is very important because of its potential industrial uses. Among these uses, it stands out the capacity to hydrolyze raffinose oligosaccharides (RO), which are the main responsible factors for gastrointestinal disorders related with the ingestion of soybean-derived products. The intestinal mucous membrane of humans and monogastric animals lacks the α-galactosidase enzyme, which is essential for the hydrolysis of these oligosaccharides. The reduction of these soybeanderived sugars can therefore improve the nutritional quality of these foods. The objective of this work was to produce, purify and characterize α-galactosidases from Aspergillus terreus, evaluating its hydrolytic capacity on raffinose oligosaccharides (RO) found in soybean products. The fungus Aspergillus terreus grew in mineral liquid medium containing wheat bran as carbon source for 168 hours at 28° C. Gel-filtration, hydrophobic interaction, ion exchange and affinity chromatographies were used to obtain purified α-galactosidase E1 and partially purified E2. Native gel was also used as the last stage of α-galactosidase E1 purification. Maximum activities of α-galactosidases E1 and E2 were detected at pH 5 and 5.5 and at the temperatures 60 and 50 oC, respectively. Enzyme α-galactosidase E1 maintained 90% of its initial activity when pre-incubated for 12 hours at 55 °C, whereas α-galactosidase E2 maintained only 5% of its activity when pre-incubated in the same conditions. KM values for ρNPαGal, mellibiose, stachyose and raffinose for α-galactosidase E1 were 0.66, 1.92, 10.94 and 27.93 mM, respectively, whereas for α- galactosidase E2 the KM for ρNPαGal and mellibiose was 0.20 and 20.14 mM, respectively. The α-galactosidases showed specificity for galactose at α position, hydrolyzing synthetic ρNPαGal substrate, mellibiose, stachyose and raffinose. Copper sulfate, mercury chloride, silver nitrate and SDS totally inactivated the two enzymes, whereas zinc sulfate totally inactivated α-galactosidase E1 and reduced α-galactosidase E2 activity by 48%. The α-galactosidase E2 in the presence of ρNPαGal substrate underwent competitive inhibition by galactose (Ki 0.76 mM). Activation energy of ρNPαGal substrate was found to be 51.17 and 36.71 kJ/mol for α-galactosidases E1 and E2, respectively. The mellibiose substrate had activation energy for α- galactosidase E1 and E2 of 53.83 and 39.83 kJ/mol respectively. Substrates raffinose and stachyose had activation energy of 55.18 and 42.81 kJ/mol respectively, for α-galactosidase E1. The treatments of defatted soybean extract with α-galactosidases E1 and E2 resulted in 100% reduction in stachyose after 12 hours of incubation, however raffinose still remained 24% higher than the initial level, which can be explained by the fact that this sugar is one of the products of stachyose hydrolysis, causing therefore its accumulation. After 12 hours of incubation of α-galactosidase E2 with the defatted extract, 18 and 25% of raffinose and stachyose were hydrolyzed, respectively. Therefore, it can be concluded that α- galactosidase E1 can be preferably used to reduce RO, being indicated for the industrial processing of these sugars.