The DAO1 gene of the yeast Trigonopsis variabilis encoding a D-amino acid oxidase (DAAO) has been cloned, sequenced, and overexpressed in Escherichia coli once the intron, which interrupts the reading frame, was eliminated by polymerase chain reaction mutagenesis. Moreover, to facilitate the purification of DAAO, a fully active tagged enzyme was constructed by engineering a six histidine tail in the N-terminal region of the protein. Unexpectedly, the resulting His-DAAO could not be purified by metal-chelate chromatography by using supports containing copper or zinc since the adsorption process inactivates the enzyme that was so strongly bound to these supports that it could be eluted only after boiling the matrix with 4% sodium dodecyl sulfate. However, we were able to purify the enzyme from a crude extract in a single step by using a tailor-made metal chelate support containing a very low density of cobalt ligands. Interestingly, the enzyme bound to this support remained active, opening a new scenario to investigate the design of an industrial process based on its immobilization in this support. This is the first time that this important industrial enzyme has been successfully modified by protein engineering to facilitate its downstream processing. Therefore, our biotechnological approach not only provides the tools to develop more efficient industrial processes, such as the production of 7-amino cephalosporanic acid from cephalosporin C, by using a highly active DAAO preparation, free from undesirable contaminant enzymatic activities, but also illustrates the importance of a careful design of the metal chelate support for optimizing the purification of His-tagged proteins.