Dissociation and reconstitution studies of a broad substrate specific multimeric alcohol oxidase protein produced by Aspergillus terreus.

A multimeric alcohol oxidase from Aspergillus terreus was dissociated and simultaneously deflavinated into catalytically inactive FAD-free subunits when incubated with 0.74 M beta-mercaptoethanol (beta-ME) for 8 h at 4 degrees C. This dissociation process had traversed through two FAD-associated intermediate proteins, between these one of them showed the enzyme activity. On removal of beta-ME, the multimeric apoprotein was regenerated, which was, however, catalytically inactive. Reactivation of the FAD supplemented apoprotein was accomplished only after incubating with the substrate. This catalytic reactivation was a slow process as evident from the prolonged FAD emission quenching. The dissociation and re-association phenomena were demonstrated by using dynamic light scattering, size exclusion chromatographic, confocal laser scan microscopic and native PAGE analyses. The solvent effect caused by the high concentration of beta-ME is attributed to the observed dissociation and linked deflavination of these multimeric alcohol oxidase protein particles.