Research On Prokaryotic Expression And Characterization Of The CMY-39Ampler Class C β-Lactamase

Objective： To examine drug sensitivity and kinetic parameter of a new subtypeCMY-39Ampler class C β-Lactamase.Methods:(1) Citrobacter freundii (Strain NO.90757) was identified by genic type. ItsAmpC production was detected by drug sensitive test using cefoxitin disc andthree-dimensional test of AmpC.(2) The PCR primers of CMY-39gene were designed according to the sequencein GenBank. The entire CMY-39encoding gene was amplified from the genomeof clinical isolated Citrobacter freundii (Strain NO.90757) by PCR andsequenced after ligated with PGEM-T vector.(3) CMY-39gene was cloned into the PET32a (+) vector. The recombinantplasmid was transformed into E.coli BL21(DE3). Expression of CMY-39in18℃was confirmed by SDS-PAGE electrophoresis.(4) Antibiotic susceptibility of recombinant strain and Strain (NO.90757)were tested by VITEK-2. Crude β-lactamase extracted from recombinant strain.The expressed protein was detected by three-dimensional test of AmpC andWestern blotting. The stability and kinetic parameters ofβ-lactamasehydrolysis about the recombinant strain and Strain NO.90757were examined.Results:(1) Strain NO.90757is identified to be Citrobacterfreundii, which couldproduce β-lactamase AmpC. This AmpC β-lactamase belonged to CMY-39. The 1146bp DNA fragment of CMY-39encoding gene was amplified from Strain(NO.90757) by PCR, and amino acid sequence analysis showed the target genewas99%homologous to CMY-39in GenBank. This CMY β-lactamase is a novelCMY-39AmpC β-lactamase, and its accession number is HM565135.(2) Prokaryotic recombinant plasmid PET32a (+)/CMY-39has been constructedsuccessfully. It could be cut into two fragments with1146bp and5901bp byrestriction endonuclease XhoⅠand EcoRⅠ.(3) After being induced with18℃overnight，a60KD recombinant infusionprotein of CMY-39was expressed in the PET32a(+)-BL21(DE3) system.(4) Antibiotic susceptibility results showed that the recombinant strain keepthe characteristic of the drug resistance. It is resistant to cephamycins,the first and second generation cephalosporin, and susceptible to the thirdand fourth generation cephalosporin, aminoglycoside and carbapenem. It couldnot be restrained by clavulanic acid.(5) The value of kinetic parameters with β-lactamase showed that the Km valueof recombinant strain is increased in different degree compared with StrainNO.90757. It indicated that the affinity of antibiotic to enzyme is reduced.Conclusions：(1) The recombinant protein of AmpC belongs to a novel subtype of CMY-39andits accession number is HM565135.(2) It is successful prokaryotic expression in the PET32a/BL21(DE3) system.(3) Compared with Strain NO.90757, the susceptibility and stability ofrecombinant strain to β-lactamase are increased, affinity is reduced.