title: Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides.
abstract: An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg⁻¹. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4-50 °C and at a pH range of 2-5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.
authors.affiliation: State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, 100193, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Du
authors.forename: Qin
authors.initials: Q
authors.lastname: Liu
authors.forename: Hexiang
authors.initials: H
authors.lastname: Wang
authors.forename: TziBin
authors.initials: T
authors.lastname: Ng
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D005690:Galactose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism*; D011887:Raffinose / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013696:Temperature; D055454:Trametes / Q000201:enzymology*; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D009844:Oligosaccharides; D013395:Sucrose; D000519:alpha-Galactosidase; D011887:Raffinose; D005690:Galactose