Purification and caracterization of α-galactosidases from fungus Penicillium griseoroseum to application in hydrolysis of ologosaccharides in soybean products / Purificação e caracterização de α-galactosidases do fungo Penicillium griseoroseum para utilização na hidrólise de oligossacarídeos de rafinose em derivados de soja

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

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.

ASSUNTO(S)

soja ologosaccharides alpha-galactosidases soybean alfa-galactosidases oligossacarídeos bioquimica

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