青霉素酶基因异源表达及该酶分解牛奶中残留青霉素的研究

Study on Heterologous Expression of Penicillinase Gene and the Penicillinase Degrading Residual Penicillin in Milk

摘要:

以获得大量青霉素酶并将其用于分解牛奶中残留青霉素为目的, 通过PCR方法从蜡样芽孢杆菌ATCC10987基因组中获得了青霉素酶基因, 将该基因克隆至表达载体pET28a(+)中, 并转化到E. coli BL21中; 在IPTG诱导下对目的蛋白进行SDS-PAGE和酶活分析, 结果显示最大酶活力可达到480.0 U/mL; 利用Ni2+亲合层析柱纯化目的蛋白, 纯化后的目的蛋白纯度超过90%; 采用高碘酸钠氧化法制备固定化的青霉素酶, 并利用该固定化酶将牛奶(含0.5 U青霉素G/mL)中的青霉素分解到浓度小于4 ppb程度。

关键词:青霉素酶, 异源表达, 纯化, 固定化青霉素酶

Abstract:

To obtain a number of penicillinases and degrade penicillin in milk by using the penicillinases, the gene encoding penicillinase was amplified by PCR from Bacillus cereus ATCC10987, cloned into pET28a(+), transformed into E. coli BL21; analysis of SDS-PAGE and penicillinase activity of the recombinant protein were done under induction of IPTG and the result showed that the maximum penicillinase activity reached 480 U/mL; the purity of penicillinase purified by Ni2+ Purification System was more than 90%; the immobilized penicillinases were obtained by sodium periodate method and the residual quantity of penicillin in milk(containing 0.5 U penicillin G/mL) was less than 4 ppb after degraded by the immobilized penicillinase.

Key words:Penicillinase, Heterologous expression, Purification, Immobilized penicillinase