M Beta Ls And Purification Of Vanx Representation And The Synthesis Of New Fluorescent Vancomycin And Drug-resistant Bacteria Inhibition

The overuse of antibiotics in agriculture, livestock and clinic has put tremendous pressure on the bacterial strains, which resulted in the emergence of a large number of antibiotic-resistant bacteria. However, continuous development of new antibiotics could not cope with the increasingly serious bacterial drug resistance. In order to combat the antibiotic resistant in bacteria, much attention has been focused on the characteristics of the target enzyme in the antibiotic-resistant bacteria, with the information gained which are useful for the rational design and synthesis of clinically useful inhibitors of the enzymes. And the alternative treatment approaches against antibiotic-resistant bacteria is still highly desirable.1. A simple and effective salting-out method was developed for purification of the matallo-β-lactamase CcrA from Bacteroides fragilis based on the plasmid pMSZ02, in which the crude protein secreted into growth medium was precipitated by making80%sulfate saturation of the medium, and purified with Q-Sepharose to offer pure CcrA with yield of20mg per litter medium. Dependence of the amount of protein precipitation on sulfate saturation was investigated, which shown that more than80%sulfate saturation makes the maximum protein precipitated. The purified CcrA was evaluated by steady-state kinetics using Penicillin G and Cephalothin V as substrates to give the Km values of68±2and17±2μM and kcat values of63±1and102±3s-1, respectively, which confirm, by comparison with the data of the protein from literature method, that the salting-out method is viable, it is useful for the purification of other proteins secreted into growth medium.2. VanX is a Zn(II)-containing metalloenzyme that is required for high-level vancomycin resistance in bacteria. The overexpression and purification of VanX were successfully achieved with the plasmid pIADL14. The purified VanX was characterized by MALDI-TOF mass spectrum and steady-state kinetics, the results indicated that this enzyme with molecular weight of23539.16exhibited Km value of0.52±0.05mM and kcat value of为6.19±0.14s-1. Based on the structure of D-Ala-D-Ala, a series of phosphonamidate and phosphonate inhibitors of VanX has been designed and synthesized. The inhibition study indicated that the phosphonamidate compound1a and phosphonate compound2a exhibited the highest activities against VanX with IC50value of0.39±0.02and0.48±0.02mM, respectively. The MIC value of the combination of norvancomycin and phosphonamidate la, phosphonate2a against Staphylococcus aureus decreased8-fold and4-fold, respectively, as well as MRSA2decreased1-fold. In addition, D-Ala-D-Ala hydrolysis catalyzed by VanX was investigated with the microcalorimetry. The activation entropy of the hydrolysis reaction is high, while the activation energy and the activation enthalpy are small, which indicate that the D-Ala-D-Ala hydrolysis with VanX is easy, exothermic and spontaneous reaction in the temperature range of293.15-308.15K3. In order to accumulate photosensitizers on the cell walls of Gram-positive antibiotic-resistant bacterial strains in labeling or/and photoinactivation of the bacteria upon light irradiation, two simple and novel conjugate norvancomycin-rhodamine B (Van-Rh), vancomycin-erythrosine (Van-Er) were synthesized, characterized and confirmed by MALDI-TOF mass spectrum. The properties of Van-Rh in photodynamic inactivation and fluorescent imaging of vancomycin-sensitive and vancomycin-resistant Enterococci (VRE) strains were investigated. The photodynamic assay indicated that Van-Rh effectively inactivated the Bacillus subtilis (ATCC6633), clinical isolates of VRE and Enterococcus faecalis (ATCC51299, Van B genotype) with inactivation rates of71,54and47%at9μM upon3min light exposure, respectively, and Van-Er exhibited much higher photodynamic antibacterial activities against MRSA. Van-Rh showed the fluorescent imaging of the B. subtilis at5μM and two VRE strains at20μM, but not the E. coli. The phototoxicity and binding affinity of rhodamine B and erythrosine were enhanced by conjugation with vancomycin as affinity ligand.4. A fluorescent probe containing the N-hydroxysuccinimidyl ester in2position was prepared from erythrosine by one-step condensation reaction. The N-hydroxysuccinimidyl erythrosine ester (ER-S) was characterized by UV-Vis and fluorescence spectrum. The photodynamic assay indicated that ER-S exhibited a little higher antitumor activity against HepG2cell as compared with erythrosine. The fluorescent imaging test indicated that ER-S could stain both the nucleus and cytoplasm, but erythrosine could only stain cytoplasm.