An alpha-galactosidase was purified from Pseudobalsamia microspora (PMG) to 1224.1-fold with a specific activity of 11,274.5 units/mg by ion-exchange chromatography and gel filtration. PMG is a monomeric protein with a molecular mass of 62 kDa as determined by SDS-PAGE and by gel filtration. Chemical modification using N-bromosuccinimide (NBS) resulted in a complete abrogation of the activity of PMG, suggesting that Trp is an amino acid essential to its activity. The activity was strongly inhibited by Hg2+, Cd2+, Cu2+, and Fe3+ ions. Three inner peptide sequences for PMG were obtained by liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis. When 4-nitrophenyl alpha-d-glucopyranoside (pNPGal) was used as substrate, the optimum pH and temperature of PMG were 5.0 and 55 A degrees C, respectively. The Michaelis constant (K (m)) value of the alpha-galactosidase on pNPGal was 0.29 mM, and the maximal velocity (V (max)) was 0.97 mu mol ml(-1) min(-1). Investigation by thin-layer chromatography (TLC) demonstrated its ability to hydrolyze raffinose and stachyose. Hence, it can be exploited in degradation of non-digestible oligosaccharides from food and feed industries.