Molecular Biological Characterization And Enzymological Study Of L1 Metallo-β-lactamase

Objective: To analyse the sequence of L1 metallo- β -lactamase encoding gene from clinical isolated stenotro-phomas maltophilia and construct the prokaryotic expression vector carrying L1 gene and expressed in E. coli BL21(DE3). To measure the pI recombinant L1 and test the stability and kinetic parametes of β -lactams hydrolysis of L1. To detect the activity of L1 in different pH and temperature.Methods:(1) The PCR primers of L1 gene were designed according to the literatures. The entire L1 encoding gene was amplified from the genome of clinical isolated stenotrophomonas maltophilia 710 by PCR and sequenced after ligated with pUCm-T vector.(2) L1 encoding gene was cloned into pET-41a(+) vector and the recombinant plasmid was transformed into E. coli BL21. Expression of L1 was comfirmed by SDS-PAGE electrophoresis.(3) Crude β -lactamase extracted from recombinant strain. The pi of recombinant L1 was determined by isoelectric focusing and influence of zink iron and inhibitor on L1 emzyme activity was observed.(4) The stability of L1 and ESBLs to different β -lactamswas compared and kinetic parametes were tested using spectr-ophotometer. The activity of LI emzyme in different pH and temperatures was observed by spectrotometer. Results:(1) The 873 bp DNA fragment of LI MBL encoding gene was amplified from strain 710 by PCR and aminoacid sequence analysis showed the target gene was 92.44% homologous to blaS of MBL LI.(2) After being inducted with IPTG, a 58 kDa recombinant fusion protein of GST and LI was expressed in the pET-41a(+) system.(3) Isoelectric focusing showed the pi of recombinant LI was 6. 7. The activity of emzyme can be inhibited by EDTA and relive by lOOMm ZnS04.(4) The recombinant LI' s capacity of hydrolyzing penicillin G is similar to that of hydrolyzing cabapenem. Aztreonam and Ceftazidime are highly stable to LI. Ph 5. 8 and 40°C were the most suitable pH and temperature for LI. Substrate inhibition for LI emzyme appeared in high substrate concentration.