title: A Fungal Alpha-Galactosidase from Pseudobalsamia microspora Capable of Degrading Raffinose Family Oligosaccharides.
abstract: 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.
authors.affiliation: State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, 100193, People's Republic of China.
authors.forename: Dongxue
authors.initials: D
authors.lastname: Yang
authors.forename: Guoting
authors.initials: G
authors.lastname: Tian
authors.forename: Fang
authors.initials: F
authors.lastname: Du
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Liyan
authors.initials: L
authors.lastname: Zhao
authors.forename: Hexiang
authors.initials: H
authors.lastname: Wang
authors.forename: Tzi Bun
authors.initials: TB
authors.lastname: Ng
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D016814:Microsporidia / Q000201:enzymology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011887:Raffinose / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007202:Indicators and Reagents; D007477:Ions; D008670:Metals; D000519:alpha-Galactosidase; D011887:Raffinose