Aminoacylase 1 (ACY1) from bovine kidney was recombinant expressed in E.coli BL21 (DE3) with the vector pETDuet. The enzymatic properties comparison between ACY1 and N-acetyl-ornithine deacetylase (NAO) were studied. Several influence factors of enzyme reaction such as pH, temperature, enzyme reaction time, surfactant and metal irons were investigated. The results indicated that the resolution of 300 mmol/L N-Ac-DL-Methionine with ACY1 needs 6 h, while NAO needs 2 h. The enzyme activity and conversion rate of NAO (1139.41 U/g and 98%) are about 1.7 times higher than ACY1 (671.24 U/g and 58.78%).