blaTEM-116型超广谱β-内酰胺酶的表达、纯化及其应用

Expression, purification and application of bla_(TEM-116) extended-spectrum β-lactamase

为大量获取低成本的TEM-116超广谱β-内酰胺酶,并分析其降解环境中β-内酰胺类抗生素残留物的可行性,本研究在Escherichia coli BL21(DE3)菌株中表达了重组TEM-116超广谱β-内酰胺酶,经亲和层析纯化、柱复性与分子筛层析纯化,得到了高纯度的目的蛋白,对其理化性质进行了分析。结果表明,重组TEM-116超广谱β-内酰胺酶的分子量、比活性分别为30kDa和476IU/mg,与天然酶性质相近。重组酶在体内外对多种青霉素、头孢菌素类药物均具有较高降解效率:10IU酶可清除1L发酵液中7000mg的青霉素G;320IU酶可清除1L尿液中各200mg的青霉素G、氨苄青霉素和头孢唑林混合抗生素;1.0～2.5IU的酶可在4℃～37℃温度范围内清除1L牛奶中80U的青霉素G;2.0×104～2.3×104IU/(kg·bw)的酶能够清除小鼠体内8.0×104～9.1×104μg/(kg·bw)的青霉素G。

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 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℃ to 37℃. Furthermore, the recombinant enzyme used at 2.0×10^4–2.3×10^4 IU/（kg·bw） （body weight） eliminated 8.0×10^4–9.1×10^4 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.