Purification and some properties of L-fucose dehydrogenase from Agrobacterium radiobacter and its application to the assay of bound-fucose in glycoconjugates.

L-Fucose dehydrogenase was found in the cell extract of Agrobacterium radiobacter and purified to homogeneity about 480-fold with 16% recovery. The molecular weight of the enzyme was approx. 64,000. The enzyme was active in the neutral pH range, unlike other L-fucose or D-arabinose dehydrogenases which are active only in the alkaline pH range. Using this enzyme and alpha-L-fucosidase F-I of Bacillus circulans (Tsuji, Y., Yamamoto, K., Tochikura, T., Seno, T., Ohkubo, Y. and Yamaguchi, H. (1990) J. Biochem. 107, 324-330) simultaneously, we developed a new coupled enzymatic method in a single buffer system for determining bound-fucose in biological materials. The fucose released by alpha-L-fucosidase F-I was oxidized with L-fucose dehydrogenase in the presence of NAD+, and the NADH formed was measured by absorbance of ultraviolet or utilized to generate color in a reaction involving CuSO4 and neocuproine. Using these methods, bound-fucose in various oligosaccharides and proteins such as lacto-N-fucopentaoses and porcine gastric mucin were quantitated within 15 min.