Purification and characterization of α-l-fucosidases from Streptomyces sp. OH11242

Abstract

α-l-Fucosidases were found in the culture fluid of Streptomyces sp. OH11242 grown with porcine gastric mucin (PGM) as the sole carbon source. The α-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 α-l-fucoside (Fucα-O-PNP). In contrast, Fase-II cleaved fucose from Fucα-O-PNP, but not fucose-containing oligosaccharides on PGM. Fase-I also hydrolyzed the α1–2 fucosidic linkage in various oligosaccharides, but not α1–3 and α1–4 fucosidic linkages. Fase-II was separated into two fractions, Fase-IIa and -IIb by Mono Q chromatography, Fase-IIb hydrolyzed α1–3 and α1–4 fucosidic linkages, but not α1–2 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 59 000 and 76 000 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.