Abstract
An alpha-galactosidase was purified from Pseudobalsamia microspora (PMG) to 1224.1-fold with a specific activity of 11,274.5units/mg by ion-exchange chromatography and gel filtration. PMG is a monomeric protein with a molecular mass of 62kDa 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 0.29mM, and the maximal velocity (V max) was 0.97μmolml(-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.
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