Purification and characterization of an alpha-L-fucosidase from Pomacea canaliculata.
An alpha-L-fucosidase (EC 3.2.1.51) was isolated from the hepatopancreas of Pomacea canaliculata. The enzyme was purified 285-fold from the crude enzyme extract by procedures involving first heat treatment, ammonium sulfate fractionation, second heat treatment, and chromatography on DEAE-Sepharose, hydroxylapatite, and L-fucosylamine-CH-Sepharose. When assayed by using p-nitrophenyl glycosides as substrates, the final preparation was free from other glycosidase activities and gave a single protein band which corresponded to alpha-L-fucosidase activity on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be 260,000 by Sephacryl S-300 column chromatography. The enzyme has two optimum pH values, 2.5 and 5.0, and the apparent Km value and the maximum velocity for p-nitrophenyl alpha-L-fucoside at both pH were calculated to be 0.45 mM and 1.46 mumol/min/mg of protein, respectively. The enzyme was shown to hydrolyze the Fuc alpha 1-->2Gal, the Fuc alpha 1-->4GlcNAc, and the Fuc alpha 1-->6GlcNAc linkages, but hardly acts on the Fuc alpha 1-->3GlcNAc linkage in various oligosaccharides.
Endo
T
T
Department of Biochemistry, University of Tokyo, Japan.
Tsukada
T
T
Hiraiwa
M
M
Uda
Y
Y
Kobata
A
A
D000645:Ammonium Sulfate; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002845:Chromatography; D005590:Fractional Precipitation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D012908:Snails / Q000201:enzymology*; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D005644:alpha-L-Fucosidase; D000645:Ammonium Sulfate