A Fungal Alpha-Galactosidase from Pseudobalsamia microspora Capable of Degrading Raffinose Family Oligosaccharides

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 Hg 2+ , Cd 2+ , Cu 2+ , and Fe 3+ ions. Three inner peptide sequences for PMG were obtained by liquid chromatography–tandem mass spectrometry (LC–MS–MS) analysis. When 4-nitrophenyl α- d -glucopyranoside (pNPGal) was used as substrate, the optimum pH and temperature of PMG were 5.0 and 55 °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 μmol 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.