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

Abstract

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.