PURIFICATION AND PROPERTIES OF FRUCTOSYLAMINE OXIDASE FROM ASPERGILLUS SP 1005

A novel enzyme that decomposes Amdori rearrangment compounds including frutosyl-e-amino acids was found in a cell-free extract of Aspergillus sp. 1005. The enzyme may be called fructosylamine oxidase and systematically, fructosylamine: oxygen oxidoreductase (defructosylating) (EC 1.5.3), due to its substrate specificity. It was purified about 75-fold to a single protein band with an overall yield of 18% from the crude extract. The purification procedure was column chromatography with Phenyl-Sepharose CL-4B and DEAE-Sephadex A-50 and gel filtration using a Sephadex G-200 column. The molecular weight of the enzyme was about 83,000 by gel filtration and 43,000 by SDS-PAGE. The prosthetic group was non-covalently bound FAD. Isoelectric point and optimum pH were 6.8 and 7.7, respectivitly. Fructosyl-derivates from alpha-L-amino acids showed high susceptibility to the enzyme, and those from e-amino acids and alpha-D-amino acids were also oxidized at a diminished rate. By the enzyme reaction under atmospheric conditions with fructosyl-glycine, glucosone, glycine, and hydrogen peroxide were formed. The configuration of D-fructose in the Amadori compounds was indispensable to the enzyme reaction. The apparent Km for fructosyl-glycine, fructosyl-beta-alanine, and fructosyl-methylamine were 2.2, 5.9, and 220 mM, respectively. The enzyme activity was inhibited by Hg2+, Cd2+, Ni2+, Cu2+, Co2+, NaN3, and p-CMB.