Bacteria Resistant Target Protein Metal - Beta Lactamase Imis Expression And Purification, Characterization And Inhibition Of Research

Bacterial resistance to β-lactam-containing antibiotics is accomplished by production of the metallo-β-lactamases (MpLs), which hydrolyze the β-lactam ring of β-lactam-containing antibiotics, lossing the activity of β-lactams. Because of the broad substrate and there are no effective inhibitor in treating bacterial resistance that produce by MβLs. Therefore, so much extensive attention had paid to MβLs.MpLs have been grouped into3distinct classes, B1,B2and B3. While extensive information is known about Bl and B3MpLs, relatively little is known about the B2enzyme. In an effort to better understand the B2enzyme, the MpLs ImiS was over-expressed in BL21(DE3) E. coli and purified by FPLC. The yield is about5mg per liter. MALDI-TOF MS showed that the enzyme have a molecular mass of25234.49. In this paper, the metal free ImiS (apo-ImiS) was also prepared. The ImiS and apo-ImiS were characterized by using metal analyses, fluorescence spectroscopy, CD spectroscopy, Zeta-potential and effective diameter.In an effort to better understand the reaction of imipenem hydrolysis by ImiS, the thermodynamic parameters of imipenem hydrolysis catalyzed by metallo-β-lactamase ImiS were determined by microcalorimetric method. The values of activation free energy△G≠θ are86.40,87.54,88.77and89.84kJ·mol-1at293.15,298.15,303.15and308.15K, respectively; the rate constant k are2.4558,2.8549,3.1917and3.7789×10-3s-1at293.15,298.15,303.15and308.15K, respectively; activation enthalpy△H≠θ is18.59kJ·mol-1; activation entropy△S≠θ is-231.34J·mol-1·K-1; apparent activation energy E is21.0843kJ·mol-1; and the reaction order is1.5.In an effort to better understand the role of ions in ImiS, the calorimetric titration were performed at298.15K. The activation free energy△G≠θ of Zn (Ⅱ) and Co (Ⅱ) binding to apo-ImiS at298.15K with values of92.948and93.908kJ·mol-1.The hydrolysis product which carried out on ImiS using imipenem as the substrate has some antibacterial activity. To deeply investigate the substrate inhibitor, the product was performed by ImiS hydrolysis imipenem completely, purified by HPLC. Liquid chromatography-mass spectrometry (LC-MS) showed that the compound has a mass of316.0827. UV-Vis spectra and fluorescence apectra were used to monitoring the catalyzed hydrolysis process. From the two spectras, the product is clearly marked off from imipenem. The antibacterial activity of the hydrolyzate was measured by the MIC and the inhibition constant Ki. Hydrolysis products have a certain inhibitory effect of ESBL E. coli, MRSA1, MRSA2, CcrA, ImiS, and L1. The kinetic results indicated that the product is a noncompetitive inhibitor. The values of Ki are118.2,41.56and49.87μM for CcrA, ImiS and L1, respectively. The structure of the product could be one of the specific modes for new metallo-β-lactamases inhibitor.