Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302

Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylam ine showed a single amine oxidase activity band in a developed polyacrylami de gel that weakly cross-reacted with the antibody against a copper/topa qu inone-containing amine oxidase (AO-II) from the same strain induced by n-bu tylamine. Since the organism cannot grow on methylamine and the already kno wn quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The e nzyme was separated and purified from the already known two quinoprotein am ine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S-2 0,w(0) of 6.5s, The molecular mass of 133 kDa estimated by gel chromatograp hy and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the en zyme. The purified enzyme was pink in color with an absorption maximum at 4 94 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylam ine, but not benzylamine, histamine, or tyramine, favorite substrates for t he already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quino ne cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining c onfirmed the presence of a quinone cofactor. pH-dependent shift of the abso rption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquin one. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organ ism.