Wild Type And The Reorganization Of Metallo-¦Â-acid Aminotransferase L1 Preparation, Spectral Characterization And Kinetic Studies

Metal p-lactamases (MpLs) including NDM-1 are triggering a bio-medical crisis, there were no any response. With the plasmid pET26b(+)Ll expression in Escherichia coli and purified B3 subgroup of metalβ-lactamase representative Ll, to study the molecular biology, mechanism and the role of different metal ions in the active sites, for the development of new enzyme inhibitors and thus provide a basis for dealing with resistant bacteria. In this paper, the following results have been got.①The plasmid containingβ-lactamase Ll gene was transferred to and expressed in Escherichia coli BL21 (DE3) at 37℃, PH 7.5, IPTG induction of protein production, cell grow for 3-14 hours,4 ml/min flow rate of elution on FPLC, SDS-PAGE to confirm the purified Ll, the yield was 10-15mg per liter of cell culture medium.②The Co(Ⅱ) substituted metallo-p-lactamase Ll (Co(Ⅱ)-Ll) was prepared successfully, and kinetic parameters of Co(Ⅱ)-Ll and wild-type Ll (WT-Ll) catalyzing three group of nine antibiotics hydrolysis were determined, and relative reactions were characterized by fluorescence, UV-Vis and CD spectra. The kinetic results indicate that the catalyzing activity of WT-L1 catalyzing carbapenems, penicillins and cephalosporins hydrolysis is strong, medium and weak, respectively, and the activities of Co(Ⅱ)-Ll to these antibiotics are lower than the WT-Ll with 2.6, 1.4 and 4.8 folds respectively.UV-Vis characterization results indicated that Co (Ⅱ)-Ll has absorption bands at 340 nm and between 500-600 nm, fluorescence spectral results show that, Co (Ⅱ)-Ll in the luminescence intensity and emission wavelength than the Apo-Ll significantly Change, and circular dichroism results show that the secondary structure Co (Ⅱ)-Ll has changes.③The first rare earth recombinant protein La(Ⅲ)-, Ce(Ⅲ)-, Pr(Ⅲ)-and Nd (Ⅲ)-Ll were prepared successfully and characterized kinetically and with ICP, fluorescence and CD spectra to explore its activity point the role of metal ions and the catalytic activity to different antibiotics. ICP test results show that, in addition to Nd (Ⅲ)-Ll, the rare earth ion contents in the recombinant protein are about 1.0±0.3/per protein molecule, which is agreement with the results from fluorescence and CD spectral characterization. CD characterization showed that the active site metal ions are replaced by rare earth ions does not cause changes of protein secondary structure, and steady-state kinetic results show that recombinant proteins show different catalytic activity to different classes of antibiotics, and the activity is 3 to 4 folds higher than the wild-type Ll.