D-Amino acid oxidase catalyzes one of the key steps in the production of semisynthetic cephalosporins. We expressed and purified recombinant Rhodolorula gracilis D-amino acid oxidase with C-terminal his-tags. This engineered enzyme was immobilized onto Ni+2=chelated nitrilotriacetic acid magnetic beads through the interaction between his-tag and Ni2+. The kinetic constants, storage properties, and the reusability of the immobilized D-amino acid oxidase were determined. The effects of temperature, pH, and hydrogen peroxide on the activity of immobilized D-amino acid oxidase were also studied. The highest activity recovery was 75%. Thermal stability was improved after immobilization; the relative activity of the immobilized enzyme was 56% whereas the free enzyme was completely inactivated after incubation at 50 degrees C for I It. In the presence of 10 mM hydrogen peroxide, the immobilized enzyme did not show a rapid loss of activity during the first 2 h of incubation, which was observed in the case of the free enzyme; the residual activity of the immobilized enzyme after 9 h was 72% compared with 22% of the free form. The long-term storage stability was improved; the residual activity of the immobilized enzyme was 74% compared with 20% of the free enzyme when stored at room temperature for 10 d. The immobilized form retained 37% of its initial activity after 20 consecutive reaction cycles.