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D-amino acid oxidase from Trigonopsis variabilis (TvDAAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids using molecular oxygen as a co-substrate. The complete catalytic cycle of TvDAAO consists of two half reactions, the conversion of the D-amino acid into the corresponding 2-oxoacid and ammonia, and the formation of H2O2 as O2 reduction product. The main industrial use of TvDAAO is the production of 7-amino cephalosporanic acid, a precursor of cephalosporin C derivatives showing antibacterial activity. The relaxed tolerance of TvDAAO towards structural changes in the side chain of the D-amino acid substrate makes the enzyme and interesting and versatile biocatalyst for the resolution of racemic mixtures of amino acids and for other applications. A drawback of TvDAAO for biotechnological use is the limited half-life of the enzyme activity under the operational conditions. Continuous supply of the oxygen co-substrate through aeration and generation of a reactive peroxide species in solution might trigger the inactivation. After decades of research on TvDAAO and related DAAOs from other sources, it is clear that numerous factors can influence DAAO stability: dissociation of subunits ; partial unfolding ; chemical modifications ; loss of the FAD cofactor. However, there appears to be no consensus about the relative importance of these factors in respect to determining the operational half-life time of TvDAAO activity in soluble or immobilised enzyme preparations. We have examined the stability of a technical-grade soluble TvDAAO at pH 8 using and oxygen independent assay for the activity and find a time course of inactivation that suggests a complex mechanism of denaturation. To further investigate the denaturation, TvDAAO has been purified to apparent homogeneity and the inactivation of pure components has been studied using electrospray tandem MS and other probes of protein integrity and conformation. The results show that control of the correct oligomeric state of TvDAAO is an essential feature of the stabilization of the enzyme activity, and prevention of thiol deactivation and exposure of hydrophobic protein surface could contribute to this effect.