ISOLATION, PURIFICATION, AND CHARACTERIZATION OF AMADORIASE ISOENZYMES (FRUCTOSYL AMINE-OXYGEN OXIDOREDUCTASE EC-1.5.3) FROM ASPERGILLUS SP

Four ''amadoriase'' enzyme fractions, which oxidatively degrade glycat ed low molecular weight amines and amino acids under formation of hydr ogen peroxide and glucosone, were isolated from an Aspergillus sp. soi l strain selected on fructosyl adamantanamine as sole carbon source. T he enzymes were purified to homogeneity using a combination of ion exc hange, hydroxyapatite, gel filtration, and Mono & column chromatograph y. Molecular masses of amadoriase enzymes Ia, Ib, and Ic were 51 kDa, and 49 kDa for amadoriase II. Apparent kinetic constants for N-epsilon -fructosyl N-alpha-t-butoxycarbonyl lysine and fructosyl adamantanamin e were almost identical for enzymes Ia, Ib, and Ic, but corresponding values for enzyme II were significantly different. FAD was identified in all enzymes based on its typical absorption spectrum. N terminal se quence was identical for enzymes Ia and Ib e-Leu-Ser-Thr-Glu-Ser-Ser-I le-Ile-Val-Ile-Gly-Ala- Gly-Thr-Trp-Gly-) and Ic except that the first 5 amino acids were truncated. The sequence of enzyme II was different l-Thr-Lys-Ser-Ser-Ser-Leu-Leu-Ile-Val-Gly-Ala-Gly- Thr-Trp-Gly-Thr-Se r-Thr-). All enzymes had the FAD cofactor-binding consensus sequence G ly-X-Gly-X-X-Gly within the N-terminal sequence. In summary, these dat a show the presence of two distinct amadoriase enzymes in the Aspergil lus sp. soil strain selected on fructosyl adamantanamine and induced b y fructosyl propylamine. In contrast to previous described enzymes, th ese novel amadoriase enzymes can deglycate both glycated amines and am ino acids.