[Expression, purification and application of bla(TEM-116) extended-spectrum beta-lactamase].
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 Ni(2+)-NTA affinity and gel filtration chromatography through subcloning the bla(TEM-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 degrees C to 37 degrees C. Furthermore, the recombinant enzyme used at 2.0x10(4)-2.3x10(4) IU/(kg bw) (body weight) eliminated 8.0x10(4)-9.1x10(4) microg/(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.
Wang
Zhen
Z
Institute of Microecology, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, China.
Zheng
Ying
Y
Fan
Quanshui
Q
Chen
Xiushu
X
Lu
Jianxin
J
D000818:Animals; D002511:Cephalosporins / Q000037:antagonists & inhibitors; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D010406:Penicillins / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D002511:Cephalosporins; D010406:Penicillins; D011994:Recombinant Proteins; D001618:beta-Lactamases