Purificação e caracterização de uma α-galactosidase em sementes de Tachigali multijuga e clonagem parcial do gene da estaquiose sintase de soja

• Ano de defesa: 2007
• Autor(a) principal: Fialho, Lílian da Silva
• Orientador(a): Rezende, Sebastião Tavares de
• Banca de defesa: Castro, Ieso de Miranda , Fietto, Luciano Gomes , Oliveira, Luiz Orlando de , Borges, Eduardo Euclydes de Lima e
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: Alfa-galactosidase; Galactooligossacarídeos; Estaquiose sintase; Soja
• Palavras-chave em Inglês: Alfa-galactosidase; Galactooligosaccharides; Stachyose synthase; Soybean
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
• Link de acesso: http://locus.ufv.br/handle/123456789/342

Because of its high nutritional value, soybean is worldwide consumed in the forms of oil, margarines, texturized protein and hydrosoluble extract. The hydrosoluble extract, known as soymilk, is also an alternative to dairy food for many lactose-intolerant people. However, the presence of galactooligosaccharides (GO) restricts the consumption of soybean and soyderived products. The intestinal mucous membrane of humans and monogastric animals lacks the α-galactosidase enzyme that is necessary for the hydrolysis of α-1,6 bonds of galactosil residues present in GO, consequently these oligosaccharides are not digested causing diarrhea, nauseas and flatulence. As part of our effort to increase the consumption of soybean and soy-derived products by humans, this work was developed with two goals: to purify and characterize one α-galactosidase from Tachigali multijuga seeds and evaluate its capacity for hydrolysing GO in soymilk; and to isolate, clone and sequence part of the gene that encodes the soybean stachyose synthase enzyme (STS), to be used for its silencing by genetic engineering techniques. The α-galactosidase enzyme was purified by acid precipitation, dialysis, ion exchange and gel filtration chromatographies. The purified enzymatic fraction was analyzed by electrophoresis in SDS-PAGE gel revealing only one protein band with estimated molecular mass of 38 kDa. Maximal α-galactosidase activity was detected at pH 5.0 5.5 and 50 ºC. The enzyme was stable at pH 4.5 - 7.0 at 40 ºC for 15 min, but lost its activity after incubation for 30 min at pH 7.0 at 40 ºC. About 65 % of original activity was kept after pre-incubation at 35 ºC for 200 h. Half-life of the α-galactosidase at 40 ºC was 17.6 h. The α-galactosidase showed absolute specificity for galactose linked to the α position, and it was completely inhibited by SDS, Hg2+, Cu2+ and Ag+ and partially inhibited by D-galactose and melibiose. The KM values for the hydrolysis of ρNPαGal, melibiose, raffinose and stachyose were 0.45, 5.37, 39.62 and 48.80 mM, respectively. The enzyme was inhibited competitively by galactose when ρNPαGal was used as substrate, with Ki of 2.74 mM. Activation energies estimated for ρNPαGal and raffinose were 13.86 and 4.75 kcal/mol, respectively. The treatment of defatted soybean flour with the α-galactosidase purified from Tachigali multijuga seeds resulted in 72 and 50 % of reduction in raffinose and stachyose contents, respectively, after incubation at 40 ºC for 4 h. The enzyme showed capacity to hydrolyze the locust bean gum and guar gum, suggesting that it can be used in the industry to improve gelling properties in polysaccharides. STS activity was determined during the development of soybean seed and in mature seeds. Stachyose quantification was carried out by HPLC. Stachyose content in mature soybean seeds was 4.10% and STS specific activity was 2.15 nkat/mg, using raffinose and galactinol as substrates. A fragment of the STS gene was isolated by PCR using seed cDNA in combination with degenerated primers. The analysis of the expression patterns by RT-PCR showed that the STS gene is expressed in all seed developmental stages, as well as in leaves, stem and roots. The cloning of a 983 base-pair fragment in a pGEM-T Easy vector was confirmed by sequencing. The identity of the fragment, using BLAST analysis, confirmed that the cloned sequence referred to the STS gene, which had not been isolated and sequenced in soybeans until then. This work opens perspectives for biotechnological researches aiming at the development of soybean varieties with reduced stachyose content and therefore more suitable for human consumption.