蜡状芽孢杆菌磷脂酶C基因在大肠杆菌中的异源表达

Cloning and Heterologous Expression in E. coli of Phospholipase C Gene from Bacillus cereus

利用磷脂酶C能够分解卵黄中的卵磷脂而在卵黄LB琼脂平板上产生乳白色晕圈,从本实验室微生物菌种库600株细菌中筛选出一株高产磷脂酶C(PLC)蜡状芽孢杆菌Bacillus cereus 12,以其染色体为模板扩增得到磷脂酰胆碱特异性磷脂酶C(PC-PLC)编码基因pcplc1,构建重组表达质粒pET28a(+)-pcplc1,并将重组质粒转入受体菌E.coli BL21(DE3)中,用异丙基硫代-β-D-半乳糖苷(IPTG)进行诱导表达。SDS-PAGE分析显示:重组蛋白以可溶性蛋白的形式存在于细胞破碎上清,分子质量约33kD,与预期蛋白大小一致。重组蛋白在硼砂卵黄平板上显现明显的乳白色晕圈,证明重组蛋白具有磷脂酶C活性,重组大肠杆菌构建成功。通过改变培养温度、IPTG诱导剂浓度及诱导时间等单因素试验初步优化得到摇瓶培养最佳诱导表达条件为:转接量4%、最佳诱导时机1.5h、最佳IPTG终浓度为0.2mmol/L,25℃诱导培养4h,最佳诱导条件下重组蛋白全部以可溶性蛋白形式存在,破碎上清酶活力达到(30.24±0.18)U/mL。

As phospholipase C （PLC） could hydrolyze egg yolk and produce a zone of opalescence surrounding colonies on an egg yolk agar plate, one phospholipase C producing strain of Bacillus cereus 12 was isolated from 600 strains preserved in our laoratory. Phospbatidylcholine-preferring phospholipase C （PC-PLC） gene pcplel was amplified from the isolated strain. Recombinant plasmid pET28a（＋）-peplel was constructed and transformed into E. eoli BL21（DE3）. After subsequent indution by isopropyl fl-D-l-thiogalactopyranoside （IPTG）, an approximately 33 kD protein was detected in the cell lysate supernatant by SDS-PAGE. Meanwhile, the recombinant protein showed significant PLC activity on egg yolk agar plate. Induction time, induction temperature and IPTG concentration were investigated and optimized induction conditions for peplel expression were obtained as follows： 4% of inoculum amount, 1.5 h of initial culture, 4 h of induced culture in the presence of 0.2 mmol/L of IPTG, and 25 ℃ of culture temperature. Unde the optimized conditions, maximum PLC activity in the supernatant was observed to be （30.24 ± 0.18） U/mL （crude enzyme activity）.