| Beta-lactamase is a significant cause of most of the pathogens resistance to beta-lactam antibiotics. Metallo-beta-lactamases (MBLs) have a broad spectrum of substrate hydrolysis including carbapenem antibiotics and have not been inhibited by clinically used beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam, so much attention was focused on metallo-beta-lactamases.In an effort to better understand the structure and function of the metallo-beta-lactamase, metallo-beta-lactamase ImiS was over-expressed in E. coli and purified, determined the optimal culture conditions. With SDS-PAGE and spectroscopic characterization including MALDI-TOF MS, UV-Vis and fluorescence, we find the metallo-beta-lactamase ImiS has a molecular weight of 25209.91 Da, and it has an obvious fluorescence at 330 nm.To better understand the structure and function of metallo-beta-lactamase ImiS, the kinetic parameters of ImiS hydrolyzing three groups (6 kinds) of beta-lactam antibiotics hydrolysis were determined by UV-Vis, and the effects of pH, temperature and substrate concentration on enzymatic activity were investigated. We also investigate kinetics of Marinopyrrole A as an inhibitor inhibiting ImiS to catalyze faropenem hydrolysis. The kinetic results indicated that Marinopyrrole A is a non-competitive inhibitor with a Ki of 24.9μmol/L and the optimal condition of ImiS activity is 44.8±0.8℃, pH=7.5±0.1.In order to deeply investigate mechanism and detect metallo-beta-lactamases, a novel fluorescent substrate of metallo-beta-lactamase is successfully constructed, synthesized and characterized. A series of chemical and biological-depth study was carried out to determine its biological and spectroscopic activities to metallo-beta-lactamase. The results indicate that the compound is photosensitive substrate of metallo-beta-lactamases, which possesses high efficiency, low toxicity, rapid and low detection. |
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