Low molecular weight α-galactosidase from black gram (Vigna mungo): Purification, characterization, and insights towards thermal denaturation.

A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal) from
germinating black gram (Vigna mungo) seeds was purified to homogeneity (432 U/mg
specific activity, 1542-fold purification) using ion-exchange (DEAE-cellulose, CMsepharose)
and affinity (Con-A Sepharose 4B) chromatography. VM-αGal appeared as a
single band ~ 45 kDa on SDS-PAGE and showed optimal activity at pH 5 and 550C. Hg2+
and SDS completely inhibited VM-αGal activity. The Km, Vmax and catalytic efficiency
(kcat/Km) of VM-αGal for pNPG and raffinose was found to be 0.99, 17.23 mM, 0.413 s-1
mM-1 and 1.66, 0.146 mM ml-1 min-1, 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 45-510C showed
that the enzyme 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
β-sheet content at 45-650C, and above 550C, respectively. VM-αGal improved the nutritional
quality of soymilk by hydrolyzing the flatulence-causing raffinose family oligosaccharides
[26.5% and 18.45% decrease in stachyose and raffinose, respectively).