We devised to engineer a His-tagged D-amino acid oxidase from the yeast Rhodotorula gracilis starting from a chimeric recombinant protein with six additional amino acid residues at the N-terminus (MARIRL). The His-tagged protein was successfully expressed in E. coli (approximate to 600 U/g cell paste) as a soluble protein and a fully active holoenzyme. No side-activities (as P-lactamase and catalase) were present in appreciable amounts. The His-tagged D-amino acid oxidase was purified in a single step by nickel-chelate chromatography to a specific activity of 115 U/mg protein at 25 degreesC and with a final procedure yield of 82%. A good thermal stability is shown by the engineered enzyme, with a T-m of 42 degreesC after 30 min incubation; this stability can be further increased up to a value of 63 degreesC by matrix immobilization. The production of this His-tagged chimeric D-amino acid oxidase thus provides the tool to optimise the downstream processing of the protein by decreasing the cost of the biocatalyst, generating a fully active holoenzyme, which properties can be further enhanced by covalent immobilization.