Isolation， Expression and Characterization of Enhanced Activity β-Lactamase

Abstract: We constructed bla_TEM mutant library with DNA family shuffling. A mutant (bla_TEM^m-pET28a/BL21) with improved activity was obtained with the elementary plate screening and secondary agar dilution. Minimum inhibitory concentrations (MICs) of E. coli BL21 (pET28a-bla_TEM^m) were two to four times (ampicillin)， four to eight times (cephazoline)， four to eight times (cefuroxime)， sixteen to thirty-two times (ceftazidime)， more eight times (ceftriaxone) and four to eight times (cefotaxime) higher than the wide type. Both TEMm and TEM were expressed efficiently in E. coli BL21 (DE3) but mainly present in inclusion bodies. The proteins could be refolded by ion-exchange chromatography using the urea linear gradient strategy， and were further purified with size-exclusion chromatography. The recovered proteins could reach 90% homogeneity. The enzymatic dynamics were determined， indicating that k_cat/K_m of β-lactam hydrolysis was all increased respectively， except cefotaxime. Gene analysis of bla_TEM^m indicated that there were several site mutations in TEM^m causing four amino acid substitutions， i.e. S59G， R164S， A237T and E240K. Tertiary structure of the mutant enzyme was predicated with swiss-Pdb Viewer 3.7， suggesting that all the substitutions did not influence the active centre of enzyme， but the subtle variation of the enzyme抯 spatial structure may enhance the affinity between enzyme and substrate.

CSTR: 32200.14.cjcb.2008.05.0023