Expression, purification and application of blaTEM-116 extended spectrum β-lactamase

Abstract:

To produce TEM-116 extended-spectrum-beta-lactamase (ESBL) from recombinant bacteria in a cost-effective way, we purified and renatured the recombinant TEM-116 ESBL from the inclusion bodies by Ni2+-NTA affinity and gel filtration chromatography through subcloning the blaTEM-116 into expression vector pET28a(+), transforming into Escherichia coli 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 a mix 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°C to 37°C. Furthermore, the recombinant enzyme used at 2.0×104–2.3×104 IU/(kg?bw) (body weight) eliminated 8.0×104–9.1×104 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.