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 blaTEM-116 extended spectrum β-lactamase

摘要:

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30 kDa和476 IU/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：10 IU 酶可清除1 L发酵液中7000 mg的青霉素G；320 IU酶可清除1 L尿液中各200 mg的青霉素G、氨苄青霉素和头孢唑林混合抗生素；1.0~2.5 IU的酶可在4℃~37℃温度范围内清除1 L牛奶中80 U的青霉素G；2.0×104~2.3×104 IU/(kg?bw)的酶能够清除小鼠体内8.0×104 ~9.1×104 μg/(kg?bw)的青霉素G。

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.