Abstract
L-Fucose (L-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23%. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34, 000. The optimum pH was at 9-10.5 and the isoelectric point was at pH 5.1. L-Fucose and L-galactose were effective substrates for the enzyme reaction, but D-arabinose was not so much. The anomeric requirement of the enzyme to L-fucose was the β-pyranose form, and the reaction product from L-fucose was L-fuconolactone. The hydrogen acceptor for the enzyme reaction was NADP+, and NAD+ could be substituted for it to a very small degree. Km values were 1.9 mM, 19 mM, 0.016mM, and 5.6mM for L-fucose, L-galactose, NADP+, and NAD+, respectively. The enzyme activity was strongly inhibited by Hg2+, Cd2+, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of L-fucose.
