Characterization of alpha-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides.
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 alpha-galactosidase. While the content of RO decreases during seed germination, the activity of alpha-galactosidase increases substantially. Two alpha-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 rho-nitrophenyl-alpha-D-galactopyranoside (rhoNPGal) were detected at pH 5.0-5.5 and 45-50 degrees C. Both enzymes were fairly stable at 40 degrees C, but lost most of their activities after 30 min at 50 degrees C. The K(m) values for hydrolysis of rhoNPGal 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 beta-mercaptoethanol showed discrete inhibitory effects on both enzymes.
Guimarães
V M
VM
Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70.910-900, Brazil. srezende@mail.ufv.br
de Rezende
S T
ST
Moreira
M A
MA
de Barros
E G
EG
Felix
C R
CR
D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005414:Flatulence / Q000517:prevention & control; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009844:Oligosaccharides / Q000737:chemistry*; D011887:Raffinose / Q000737:chemistry; D012639:Seeds / Q000201:enzymology; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics; D000519:alpha-Galactosidase / Q000302:isolation & purification / Q000378:metabolism*
D009844:Oligosaccharides; D000519:alpha-Galactosidase; D011887:Raffinose