Overexpression inEscherichia coliof a Recombinant ChimericRhodotorula gracilisd-Amino Acid Oxidase

This paper reports a novel expression system constructed to maximize the production inEscherichia coliofd-amino acid oxidase from the yeastRhodotorula gracilis(RgDAAO). We produced a recombinant plasmid by the insertion of the cDNA encoding for the RgDAAO into the multiple cloning site of the expression vector pT7.7 (pT7-DAAO), downstream of the T7 RNA polymerase binding site. The pT7-DAAO, which encodes a fully active fusion protein with six additional residues at the N-terminus of DAAO, was used to transform the BL21(DE3) and BL21(DE3)pLysSE. colicells. In the latter host and under optimal IPTG induction conditions, soluble and active chimeric DAAO was expressed in these cells up to 930 U/g of cell (and a fermentation yield of 2300 U/liter of fermentation broth), with a specific activity of 8.8 U/mg protein. RgDAAO represents ≈8% of the total soluble protein content of the cell.