Purification And Characterization Of NDM-1 And Real-time Detection Of Its Activity In Bacterial Live Cells And Its Inhibition

The problem of resistant antibiotic is becoming more and more serious. As the powerful weapon of the resistant antibiotic bacterial, β-lactamases is attracting much attention. And metallo-beta-lactamases is the focus of attention, because of its powerful ability of antibiotic hydrolysis. Recently, the appearance of New Delhi metallo-β-lactamase-1 (NDM-1) makes an emerging antibiotic-resistance threat. The bacteria contained NDM-1 shows a new era of antibiotic resistance because of the enzyme’s ability to hydrolyze almost all known β-lactam containing antibiotics and the rapid spread of the plasmid encoded NDM-1 gene. To fight against the drug-resistant bacteria, we need study the metallo-β-lactamase, and find new method to discover and design inhibitors, and apply a new tech to detect metallo-β-lactamases in the drug-resistant bacteria. Based on this, we carried out our research as follows:1. MβL NDM-1 was over-expressed in BL21 (DE3) E. coli, purified by FPLC, and identified by SDS-PAGE. Then NDM-1 was characterized by UV-visible spectroscopy, the metal free NDM-1 (Apo-NDM-1) and recombinant NDM-1 were also prepared and characterized by fluorescence spectroscopy. And NDM-1 biological activity were measured by cefazolin, imipenem, ampicillin. The study of eight compounds inhibiton was done.2. A UV-Vis spectroscopy method is reported that affords the ability to monitor the hydrolytic decomposition of the cephalosporins antibiotic cefazolin inside Escherichia coli cells expressing New Delhi metallo-β-lactamase-1 (NDM-1), an emerging antibiotic-resistance threat. NDM-1 activity in cells was shown in imipenem, penicillin as well as cefazolin. Cell-based UV-Vis studies also demonstrated that the hydrolysis of cefazolin in vivo was inhibited by two known NDM-1 inhibitors, D-captopril and ethylenediaminetetraacetic acid (EDTA), and a reported inhibitor. With this method, the activities of clinical bacteria in the antibiotics surrounding can also be observed directly.3. We designed and synthesized two flourescent cephalosporins. And they were tested by UV-visible spectroscopy, fluorescence spectroscopy.