Purification and characterization of a strictly specific β- d-fucosidase from Aspergillus phoenicis

Abstract

Although β- d-fucosidase (β- d-fucohydrolase, EC 3.2.1.38) has been isolated from various sources, all those enzymes were associated with a high activity of β- d-galactosidase and/or β- d-glucosidase. We have purified a specific β- d-fucosidase in electrophoretically homogeneous form from crude extracts of Aspergillus phoenicis by polyethyleneglycol 8000-phosphate buffer aqueous two-phase separation, and successive chromatography on DEAE-Sephadex A-50, hydroxyapatite, and Sephadex G-100 columns. The molecular weight of the enzyme was estimated to be 57,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 50,000 to 60,000 by gel filtration on Sephadex G-100. The enzyme showed optimum activity at pH 6.0 and 40 °C; it was stable in the pH range 5.5–6.5 and below 35 °C. The K m and the V max values for pNP-β - d-fucoside were 2.4 m m, and 12.8 μmol · min −1 · mg −1, respectively. The enzyme was strongly inhibited by sulfhydryl group reagents, p-chloromercuribenzoate, n-ethylmaleimide, and iodoacetate. It was also inhibited by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, diethyl pyrocarbonate, and N-bromosuccinimide. Thus, -SH and -COOH groups and histidyl and tryptophyl residues were essential for enzyme activity. The purified β- d-fucosidase showed high specificity toward p-nitrophenyl-β- d-fucoside. The enzyme was inhibited by d-fucose and d-fucono-γ-lactone, but not by d-galactose, d-galactono-γ-lactone, d-glucose, or d-glucono-γ-lactone; the latter compounds are specific inhibitors of β- d-galactosidase and β- d-glucosidase, respectively. Thus, this enzyme is the most strictly specific β- d-fucosidase when compared with those previously reported.