Characterization of a new alpha-L-fucosidase isolated from the marine mollusk Pecten maximus that catalyzes the hydrolysis of alpha-L-fucose from algal fucoidan (Ascophyllum nodosum).
Algal fucoidan is an alpha-L-fucose-based polysaccharide endowed with important biological properties for which the structure has not yet been fully elucidated. In an attempt to implement new enzymatic tools for structural study of this polysaccharide, we have found a fucosidase activity in the digestive glands of the common marine mollusk Pecten maximus, which is active on a fucoidan extracted from the brown algae Ascophyllum nodosum. We now report the purification and characterization of this alpha-L-fucosidase (EC 3.2.1.51). The enzyme was purified by three chromatographic steps, including an essential affinity chromatography based on the glycosidase inhibitor analog 6-amino-deoxymannojirimycin as the ligand. The purified alpha-L-fucosidase is a tetrameric glycoprotein of 200 kDa that hydrolyzes the synthetic substrate p-nitrophenyl alpha-L-fucopyranoside with a K(m) value of 650 microM. This enzyme has high catalytic activity (85 micromol x min(-1) x mg(-1)) compared with the other known fucosidases and also possesses an unusual thermal stability. The purified alpha-L-fucosidase is a retaining glycosidase. The activity of the purified fucosidase was determined on two structurally different fucoidans of the brown algae A. nodosum and Fucus vesiculosus to delineate glycosidic bond specificity. This report is to our knowledge the first demonstration of a fucosidase that can efficiently release alpha-L-fucose from fucoidan.
Berteau
Olivier
O
Laboratoire de Recherches sur les Macromolécules, UMR 7540 CNRS, Université Paris 13, avenue J.-B. Clément, 93430 Villetaneuse, France.
McCort
Isabelle
I
Goasdoué
Nicole
N
Tissot
Bérangère
B
Daniel
Régis
R
D000818:Animals; D002240:Carbohydrate Sequence; D002384:Catalysis; D002846:Chromatography, Affinity; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008974:Mollusca / Q000201:enzymology*; D019906:Nuclear Magnetic Resonance, Biomolecular; D000459:Phaeophyceae / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D011134:Polysaccharides; D005643:Fucose; C007789:fucoidan; D005644:alpha-L-Fucosidase