Purification and characterization of alpha-L-fucosidases from Streptomyces sp. OH11242.
alpha-L-Fucosidases were found in the culture fluid of Streptomyces sp. OH11242 grown with porcine gastric mucin (PGM) as the sole carbon source. The alpha-L-fucosidases were purified by ammonium sulfate precipitation followed by chromatography on Sepharose CL-4B, hydroxyapatite, Resource Q and Mono Q. Two enzyme fractions, termed Fase-I and Fase-II, were obtained, each bearing different substrate specificity. Fase-I hydrolyzed fucose residues from fucose-containing oligosaccharide chains on PGM, but not p-nitrophenyl alpha-L-fucoside (Fucalpha-O-PNP). In contrast, Fase-II cleaved fucose from Fucalpha-O-PNP, but not fucose-containing oligosaccharides on PGM. Fase-I also hydrolyzed the alpha1-2 fucosidic linkage in various oligosaccharides, but not alpha1-3 and alpha1-4 fucosidic linkages. Fase-II was separated into two fractions, Fase-IIa and -IIb by Mono Q chromatography, Fase-IIb hydrolyzed alpha1-3 and alpha1-4 fucosidic linkages, but not alpha1-2 fucosidic linkages, while Fase-IIa hydrolyzed none of them. Fase-I was purified to homogeneity by SDS-polyacrylamide gel electrophoresis, the molecular mass was estimated to be approximately 59000 and 76000 Da by SDS-PAGE and gel-permeation chromatography, respectively. The optimum pH for Fase-I activity was 5.5-6.0. These fucosidases with different substrate specificities might be useful to reveal the physiological role of fucose-containing oligosaccharides in the gastric mucins.
Goso
Y
Y
Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Japan. goso@kitasato-u.ac.jp
Ishihara
K
K
Sugawara
S
S
Hotta
K
K
D000818:Animals; D002850:Chromatography, Gel; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D005752:Gastric Mucins / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D005644:alpha-L-Fucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D017077:Culture Media, Conditioned; D005752:Gastric Mucins; D006027:Glycosides; C014125:4-nitrophenylfucoside; D005644:alpha-L-Fucosidase