The gene encoding the fructosyl-amino acid oxidase (fructosyl-alpha-L-amino acid: oxygen oxidoreductase (defructosylating); EC 1.5.3) of Corynebacterium sp. 2-4-1 was cloned and expressed in Escherichia coli. The gene consists of 1,116 nucleotides and encodes a protein of 372 amino acids with a predicted molecular mass of 39,042. The open reading frame was confirmed as the gene of the fructosyl-amino acid oxidase by comparison with the N-terminal amino acid sequence of the purified fructosyl-amino acid oxidase from Corynebacterium sp. 2-4-1. The sequence of the AMP-binding motif, GXGXXG, was found in the deduced N-terminal region. The amino acid sequence of the fructosyl-amino acid oxidase showed no similarity to that of fungal fructosyl-amino acid oxidases. In addition, substrate specificities of this fructosyl-amino acid oxidase were different from those of other fructosyl-amino acid oxidases. The fructosyl-amino acid oxidase of Corynebacterium sp. 2-4-1 is an enzyme that has unique substrate specificity and primary structure in comparison with fungal fructosyl-amino acid oxidases.
L-Fucose (L-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1 143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammoniumsulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyaerylamide 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 /?-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. Kmvalues were 1.9mM, 19mM, 0.016mM, and 5.6dim for L-fucose, lgalactose, 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.
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