Characterization of α-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides

• Published in: Phytochemistry (Oxford) 2001-09, Vol.58 (1), p.67-73
• Main Authors: Guimarães, Valéria Monteze, de Rezende, Sebastião Tavares, Moreira, Maurilio Alves, de Barros, Everaldo Gonçalves, Felix, Carlos Roberto
• Format: Article
• Language: English
• Subjects: alpha-Galactosidase - isolation & purification; alpha-Galactosidase - metabolism; Binding, Competitive; Biological and medical sciences; Chromatography, Affinity; Chromatography, Ion Exchange; Flatulence; Flatulence - prevention & control; Food industries; Fundamental and applied biological sciences. Psychology; Glycine max - enzymology; Glycine max L; Humans; Hydrolysis; Kinetics; Leguminosae; Oligosaccharides - chemistry; Raffinose - chemistry; Raffinose oligosaccharides; Research and development. New food products, dietetic foods and beverages; Seeds - enzymology; Substrate Specificity; Thermodynamics; α-Galactosidase
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/S0031-9422(01)00165-0

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by α-galactosidase. While the content of RO decreases during seed germination, the activity of α-galactosidase increases substantially. Two α-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M r of 33 kDa, and the second (P2) had two proteins with M r of 31 and 33 kDa. Maximal activities against the synthetic substrate ρ-nitrophenyl-α- d-galactopyranoside ( ρNPGal) were detected at pH 5.0–5.5 and 45–50°C. Both enzymes were fairly stable at 40°C, but lost most of their activities after 30 min at 50°C. The K m values for hydrolysis of ρNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K m values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor ( K i=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO 4, and SDS. Sucrose and β-mercaptoethanol showed discrete inhibitory effects on both enzymes. α-Galactosidases from germinating soybean seeds were purified and characterized, and tested against raffinose oligosaccharides present in soya milk.