alpha-Galactosidase of Bifidobacterium adolescentis DSM 20083

Bifidobacterium adolescentis was grown anaerobically in medium enriched wit h 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-e xclusion chromatography. Two protein bands were consistantly observed after sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Elect rophoretically homogeneous alpha-galactosidase was only obtained by electro elution. The enzyme had an apparent molecular mass of 344 kDa and 79 kDa as judged by size-exclusion chromatography and SDS-PAGE, respectively. Activi ty-staining after nondenaturing SDS-PAGE indicated an apparent molecular ma ss of 145 kDa. Thus, a tetrameric structure of the protein is suggested. Th e alpha-galactosidase showed optimal activity at pH 5.5 and 55 degrees C. L ower pH values and higher temperatures rapidly inactivated alpha-galactosid ase. The enzyme hydrolyzed specifically alpha-galactosidic linkages, and al pha-(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 w ere calculated with 0.957 mM and 4.12 mM, respectively.