Influence of detergents and pH on the isolation, purification and molecular properties of alpha-galactosidase-C2 from germinating guar (Cyamopsis tetragonolobus).

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.

α-Galactosidase from germinating guar (Cyamopsis tetragonolobus) seeds.

The changes in α-galactosidase activity in guar (Cyamopsis tetragonolobus) seeds was followed during seven days of germination. The enzyme activity was maximal on the first day of germination and gradually decreased during subsequent days. On the second day of germination the partially purified enzyme upon ion-exchange chromatography on CMSephadex C-50 was resolved into α-galactosidase-A (anionic), α-galactosidase-C1 (cationic) and α-galactosidase-C2 (cationic) and their relative proportions were 28,12 and 60%, respectively. The combined α-galactosidase C1 and C2 activities increased in the first two days of germination followed by significant decrease after the 3rd day onwards, whereas α- galactosidase-A remained fairly constant throughout the germination period, α- Galactosidase-A and C2 had different Km and Vmax values with p-nitrophenyl α-Dgalactopyranoside, raffinose and melibiose as substrates and also differed in their thermal stabilities.