Alpha-galactosidase of Bifidobacterium adolescentis DSM 20083.
Bifidobacterium adolescentis was grown anaerobically in medium enriched with alpha-D-galactosides. alpha-Galactosidase (EC 3.2.1. 22) was released from the cells by ultrasonic treatment and purified 36-fold by ultrafiltration, ammonium-sulphate precipitation, anion-exchange chromatography, and size-exclusion chromatography. Two protein bands were consistantly observed after sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretically homogeneous alpha-galactosidase was only obtained by electroelution. The enzyme had an apparent molecular mass of 344 kDa and 79 kDa as judged by size-exclusion chromatography and SDS-PAGE, respectively. Activity-staining after nondenaturing SDS-PAGE indicated an apparent molecular mass of 145 kDa. Thus, a tetrameric structure of the protein is suggested. The alpha-galactosidase showed optimal activity at pH 5.5 and 55 degrees C. Lower pH values and higher temperatures rapidly inactivated alpha-galactosidase. The enzyme hydrolyzed specifically alpha-galactosidic linkages, and alpha-(1-3)-linkages were hydrolyzed at a higher rate compared to alpha-(1-6)-linkages. Hydrolysis of galactosides followed normal saturation kinetics; KM-values for p-nitrophenyl-alpha-galactopyranoside (p-NPG) and raffinose were calculated with 0.957 mM and 4.12 mM, respectively.
Leder
S
S
Institute of Pharmacology and Toxicology, Wendlingweg, Aachen University of Technology, 52056 Aachen, Germany.
Hartmeier
W
W
Marx
S P
SP
D001644:Bifidobacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D000519:alpha-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000519:alpha-Galactosidase