The arginine deiminase (ADI, E.C 3.5.3.6) - a key enzyme of ADI pathway of Enterococcus faecium GR7 was purified to homogeneity. A sequential purification strategy involving ammonium sulfate fractionation, molecular sieve followed by Sephadex G-100 gel filtration was applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified with a fold of 16.92 and showed a final specific activity of 76.65 IU/mg with a 49.17% yield. The dimeric ADI has a molecular mass of about 94,364.929 Da, and comprises of hetrodimers of 49.1 kDa and 46.5 kDa as determined by MALDI-TOF and PAGE analysis. To assess anti-cancerous activity of ADI by MTT assay was carried out against cancer cell lines (MCF-7, Sp2/0-Ag14 and Hep-G2). Purified ADI exhibited the most profound antiproliferative activity against Hep-G2 cells; with half-maximal inhibitory concentration (IC50) of 1.95 mu g/ml. Purified ADI from E. faecium GR7 was observed to induce apoptosis in the Hep-G2 cells by DNA fragmentation assay. Our findings suggest the possibility of a future use of ADI from E. faecium GR7 as a potential anticancer drug. (C) 2015 Elsevier Inc. All rights reserved.