Cloning and expression of NDM-1 metallo-β-lactamase gene and study of the catalytic properties of the recombinant enzyme

Resumo

A gene-expression system has been designed to express the NDM-1 metallo-β-lactamase gene in E. coli cells. This system enables the synthesis of the recombinant protein in a soluble and active form. A method for the isolation and purification of the recombinant enzyme has been developed. The yield of the homogeneous protein preparation was 10–15 mg per liter of E. coli culture medium. The catalytic parameters of the recombinant NDM-1 β-lactamase were measured for ampicillin (K_m = 185 μM and k_cat = 585 s^–1) and meropenem (K_m = 85 μM and k_cat = 160 s^–1). These values correlate well with the literature data. The catalytic parameters for the chromogenic CENTA substrate (K_m = 14 μM and k_cat = 290 s^–1) were obtained for the first time.