This study aims to use neutral pH optimum arginase as the catalyst for high-efficiencyl-ornithine production.Sulfobacillus acidophilusarginase was firstly cloned and overexpressed inEscherichia coli. The purified enzyme was obtained, and the molecular mass determination showed that this arginase was a hexamer.S. acidophilusarginase possessed similarities with the other arginases such as the conserved sequences, purification behavior, and the necessity for Mn(2+)as a cofactor. The maximum enzyme activity was obtained at pH 7.5 and 70 degrees C. Thermostability and pH stability analysis showed that the arginase was stable at 30-60 degrees C and pH 7.0-8.5, respectively. The kinetic parameters suggested thatS. acidophilusarginase could efficiently hydrolyzel-arginine. Bioconversion with this neutral pH optimum arginase had the advantages of avoiding producing by-product, high molar yield, and high-level production ofl-ornithine. When the bioconversion was performed with a fed-batch strategy and a coupled-enzyme system involvingS. acidophilusarginase and Jack bean urease, the final production of 2.87 mol/L was obtained with only 1.72 mmol/Ll-arginine residue, and the molar yield was 99.9%. The highest production record suggests thatS. acidophilusarginase has a great prospect in industriall-ornithine production.