Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification and insights towards biochemical and biophysical properties.

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal-P) from germinating black gram (Vigna mungo) seeds was purified (324 U/mg specific activity, 1157-fold purification, ~45 kDa) using ion-exchange (DEAE-cellulose, CM-sepharose), gel filtration (Sephadex G-75) and affinity (Con-A Sepharose 4B) chromatography but with poor yield (0.75%). Partially purified enzyme (VM-αGal) (146.3 U/mg specific activity, 522.5-fold purification) was used for further studies. VM-αGal showed optimal activity at pH 5 and 55 °C. Hg2+ and SDS completely inhibited VM-αGal activity. The Km, Vmax and catalytic efficiency (kcat/Km) of VM-αGal for pNPG and raffinose was 0.99, 17.23 mM, 1.66, 0.146 µmol ml-1 min-1, and 0.413, 0.0026 s-1 mM-1, respectively. VM-αGal was competitively inhibited by galactose (Ki 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that VM-αGal was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in ß-sheet content at 45-65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively).