Purificação e caracterização de α-galactosidases do fungo Penicillium griseoroseum para utilização na hidrólise de oligossacarídeos de rafinose em derivados de soja

• Autor(a) principal: Falkoski, Daniel Luciano
• 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/123456789/2478
• topic: alfa-galactosidases; Soja; Oligossacarídeos; alpha-galactosidases; Soybean; Ologosaccharides; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

The present work aims to evaluate the efficiency of the purified α-galactosidases from Penicillium griseoroseum in the hydrolysis of the RO present in the free fat soy extract. Penicillium griseoroseum was cultivated in a mineral medium which contained galactomanana as a source of carbone, for 120 hours, at 28 oC. After this period, the medium was dialysed, centrifuged and used as a source of enzymes α-galactosidases. The enzymatic extracts were submitted to the chromatography in DEAE-Sepharose, pH 5.0. Adsorbed proteins were liberated with linear increasing gradient of NaCl (0-0,3 M) and two peaks containing α-galactosidase activity was detected. The enzyme detected on first peak was denominated α-Gal I and the enzyme detected on second peak was denominated α-Gal II. α-Galactosidase active fractions were pooled and concentrated by ultrafiltration cell using 10 kDa cut-off membrane. Concentrated samples were submitted in a native electrophoresis polyacrylamide gel and in the end of the electrophoresis the gel was incubated with ρ-NP-αGal solution (4 mg.mL.-1) to visualizing of the enzymes α-galactosidases. The places containing the enzymes α-galactosidases were cut off, macerated with 100 mM sodium acetate buffer (pH 5) and kept under agitation for 24 h to extracting the purified enzymes. The enzymes α-Gal I and α-Gal II got purification factors of 155 and 53 times, respectively, with a final result of 38 and 9 %, respectively. The maximum activities of the α-Gal I and α-Gal II were detected in pH 5.0 at 45 oC. The values of half-life to α-Gal I at 40 and 45 °C were 16 and 0,66 h respectively. The values of half-life to α-Gal II at 40 and 45 °C were 3,8 and 0,25 h respectively. The values of KM for ρ-NP-αGal, ο-NP-αGal, melibiose, stachyose and raffinose for the α-Gal I were 1,06, 1,31, 4,77, 19,99 e 28,74 mM, respectively, while for the α-Gal II were 0,80, 1,26, 5,10, 21,74 e 30,46 mM, respectively. The α-galactosidases presented absolute specification for galactose in α position, hydrolysing the substrates ρ-NP-αGal, ο-NP-αGal, stachyose, raffinose, melibiose. Copper sulphate, iron sulphate and silver nitrate completely deactivated the α-galactosidases from P. griseoroseum in a concentration of the 1 mM. The results of the treatments of free fat soy extract with enzymes α-Gal I and α-Gal II demonstrated a reduction of 100 % of the stachyose after 8 h of incubation at 40 oC. There were 69 and 12% of reduction of the raffinose after 8 h of incubation at 60 oC, with the enzyme α-Gal I and α-Gal II, respectively. The enzyme α-Gal I was immobilized in insoluble support (modified silica) and used in assays of hydrolysis of RO present in free fat soy extract. The immobilized enzyme hydrolysed 100 and 70% of stachyose and raffinose, respectively, after 8 h of incubation. The immobilized enzyme α-Gal I was reapplied 8 times consecutively in hydrolysis treatment without loss in its activity. Therefore, it can be observed that the α-galactosidases of P. griseoroseum were efficient in reducing the RO presents in soy derived products, and they are appropriate to industrial use in the processing of these sugars.