Expression,purification and application of bla_(TEM-116) extended-spectrum β-lactamase
blaTEM-116型超广谱β-内酰胺酶的表达、纯化及其应用

To produce TEM-116 extended-spectrum β-lactamase(ESBL)from recombinant bacteria in a cost-effective way,we purified and renatured the recombinant TEM-116 ESBL from the inclusion bodies by Ni~(2+)-NTA affinity and gel filtration chromatography through subeloning the bla_(TEM-116) into expression vector pET28a(+),transforming into Escherichia coil BL21(DE3)and inducing with IPTG.We characterized the purified protein that had the molecular weight of 30 kDa and specific activity of 476 IU/mg.The recombinant TEM-116 ESBL showed higher efficiency in eliminating penicillin and cephalosporin in vitro and in vivo.Specifically,the recombinant TEM-116 ESBL could eliminate 7000 mg penicillin G(PG)when used at 10.0 IU in 1 L fermentation medium.When used at 320.0 IU,it could also degrade amix of PG,ampicillin and cefazolin each at 200 mg in 1 L of urine.In milk,1.0-2.5 IU of the recombinant enzyme could remove 80 U/L of PG The recombinant enzyme was fully active at the temperature ranged from 4℃ to 37℃.Furthermore,the recombinant enzyme used at 2.O×x10~4-3×10~4 IU/(kg·bw)(body weight)eliminated 8.0×10~4-9.1x10~4 μg/(kg·bw)PG in mouse models in vivo.The recombinant TEM-116 ESBL has the potential as a tool enzyme in food and environmental protection to eliminate harmful residues of antibiotics.