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Purification and characterization of alpha-L-fucosidases from Streptomyces sp. OH11242
Y Goso1, K Ishihara, S Sugawara
1Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Japan. goso@kitasato-u.ac.jp
Abstract:
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.
Insights
Streptomyces sp. OH11242 produces two alpha-L-fucosidase enzymes, Fase-I and Fase-II, that cleave fucose residues from porcine gastric mucin (PGM) and synthetic substrates with distinct specificities, aiding in understanding PGM oligosaccharide roles.
Area of Science:
- Microbiology
- Enzymology
- Glycobiology
Background:
- Porcine gastric mucin (PGM) contains complex fucosylated oligosaccharides.
- Alpha-L-fucosidases are enzymes that hydrolyze alpha-L-fucose residues.
Purpose of the Study:
- To isolate and characterize alpha-L-fucosidases from Streptomyces sp. OH11242.
- To investigate the substrate specificity of these enzymes for understanding fucose-oligosaccharide roles in PGM.
Main Methods:
- Cultivation of Streptomyces sp. OH11242 with PGM as the sole carbon source.
- Enzyme purification using ammonium sulfate precipitation and multiple chromatography techniques (Sepharose CL-4B, hydroxyapatite, Resource Q, Mono Q).
- Enzyme activity assays using PGM and p-nitrophenyl alpha-L-fucoside (Fucalpha-O-PNP) as substrates.
Main Results:
- Two distinct alpha-L-fucosidase fractions, Fase-I and Fase-II, were purified.
- Fase-I hydrolyzed fucose from PGM oligosaccharides, specifically targeting alpha1-2 linkages.
- Fase-II was further separated into Fase-IIa and Fase-IIb, with Fase-IIb hydrolyzing alpha1-3 and alpha1-4 linkages from Fucalpha-O-PNP.
Conclusions:
- Streptomyces sp. OH11242 secretes alpha-L-fucosidases with differential substrate specificities.
- These enzymes, particularly Fase-I and Fase-IIb, can cleave specific fucosidic linkages found in PGM.
- The characterized fucosidases offer valuable tools for elucidating the physiological functions of fucose-containing oligosaccharides in gastric mucins.
