title: Purification and characterization of a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum.
abstract: Food ingredients containing alpha-1,6-galactoside bonds elicit gastrointestinal disturbances in monogastric animals, including humans. Pretreatment of such ingredients with alpha-galactosidase (EC 3.2.1.22) has the potential to alleviate this condition. For this purpose, a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum was purified by a combination of anion exchange and size exclusion chromatographies. The enzyme has a monomeric molecular weight of approximately 80 kDa; however, it is active as a dimer. The optimum temperature for enzyme activity is 77.5 degrees C. Approximately 84 and 88% of enzyme activity remained after 36.5 h of incubation at 70 and 65 degrees C, respectively. Optimum activity was observed at pH 8.0, with a broad range of activity from pH 5.0 to 9.0. Different transition metals had weak to strong inhibitory effects on enzyme activity. The K(m) and V(max) of the enzyme are 0.29-0.345 mM and 200-232 micromol/min/mg of protein, respectively. Importantly, enzyme activity was only slightly inhibited by 75-100 mM galactose, an end product of hydrolysis. Enzyme activity was specific for the alpha-1,6-galactosyl bond, and activity was demonstrated on melibiose and soy molasses.
authors.affiliation: Department of Food Science and Human Nutrition and Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana 61801, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: King
authors.forename: Bryan A
authors.initials: BA
authors.lastname: White
authors.forename: Hans P
authors.initials: HP
authors.lastname: Blaschek
authors.forename: Bruce M
authors.initials: BM
authors.lastname: Chassy
authors.forename: Roderick I
authors.initials: RI
authors.lastname: Mackie
authors.forename: Isaac K O
authors.initials: IK
authors.lastname: Cann
mesh_terms: D000595:Amino Acid Sequence; D003013:Clostridium / Q000201:enzymology*; D019281:Dimerization; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D000519:alpha-Galactosidase