Using three-phase partitioning followed by filtration chromatography with Sephadex G-100,an intracellular α-galactosidase from Rhizopus sp.A01 grown on soya bean dregs broth was purified to homogeneity with a 54.8-fold increase in specific activity and 27.3% recovery.The relative molecular weight of the enzyme was about 302 ku through gel filtration and 85.6 ku through SDS-polyacrylamide gel electrophoresis,suggesting that the native enzyme was a tetramer.The α-galactosidase showed high activity against p-nitrophenyl-α-d-galactopyranoside(pNPGal) but had little activity for melibiose and raffinose,and the optimal activity was observed at pH 4.5 and 55℃.The kinetic parameters of Km and kcat/ Km were 0.242 ± 0.027 mmol/L and 4.089 × 105 L /(mol.s) with pNPGal,and the rates of hydrolysis for melibiose and raffinose were 138.3 μmol /(h.mg) and 19.7 μmol /(h.mg),respectively.The enzyme activity was significantly activated by Fe2 + and Fe3 +,but strongly inhibited by Mn2 +,Cu2 +,Hg+ and Mg2 + at 5.0 mmol/L.The α-galactosidase was highly stable over pH range from 4.0 to 8.2 at 25℃,and it retained approximately 48% of the original activity after incubation for 90min at 50℃.