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New Delhi metallo-β-lactamase (NDM-1) is a new metallo-β-lactamase (MBL) that has recently emerged as a global threat because it confers bacteria with resistance to almost all clinically used β-lactam antibiotics. To determine the molecular basis of this threat, NDM-1 was purified from Escherichia coli TransB (DE3) carrying cloned blaNDM-1 gene by an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme was stable even in extremely alkaline buffer (pH 11) and reached its highest activity at a low temperature (15°C), which was different from other MBLs. The 50% inhibition concentration of EDTA against NDM-1 was 412 nM, which showed that NDM-1 was more susceptible to EDTA than other MBLs. The effects of zinc on NDM-1 differed between cephem and carbapenem complexes, but inhibition at high Zn(2+) concentration was observed for all of tested β-lactam compounds.