摘要
基于脂肪氧合酶(LOX)活性对宿主菌潜在的危害,分别共表达了超氧化物歧化酶和过氧化氢酶,以期提高铜绿假单胞菌Pseudomonas aeruginosa BBE来源的LOX在大肠杆菌Escherichia coli Rosetta(DE3)中的表达。将P.aeruginosa BBE超氧化物歧化酶基因sodB和sodM及E.coli过氧化氢酶基因katE克隆至pRSFDuet-1,分别得到表达质粒pRSF-sodB,pRSF-sodM和pRSF-katE,将上述表达质粒转化至表达LOX的重组大肠杆菌N6,得到菌株N6-B,N6-M和N6-K。在20℃和1 mmol/mL异丙基-β-D-硫代半乳糖苷(IPTG)条件下诱导70 h,N6-B、N6-M和N6-K胞内外LOX总酶活分别为21.6、28.1和7.1 U/mL,其中N6-B和N6-M较对照菌株N6(11.8 U/mL)提高了83%和138%。通过正交实验确定LOX较优的诱导表达条件为:IPTG浓度2 mmol/mL,诱导菌体浓度(OD600)2.5,诱导温度20℃。研究结果表明:共表达超氧化物歧化酶能有效促进LOX在大肠杆菌中的表达,为该酶高效异源表达研究提供了新思路。
Based on the potential hazards of lipoxygenase(LOX)activity to host strain,three antioxidases(two superoxide dismutases and a catalase)are respectively co-expressed to improve the expression of lipoxygenase expression in Escherichia coli Rosetta(DE3).The superoxide dismutase genes(sodB and sodM)from P.aeruginosa BBE and catalase gene(katE)from E.coli are cloned into expression plasmid pRSFDuet-1,yielding the plasmids pRSF-sodB,pRSF-sodM and pRSF-katE.The recombinant plasmids are transformed into N6(a LOX expressing strain generated by E.coli)to obtain strains N6-B,N6-M and N6-K,respectively.Under the induction with 1 mmol/mL ITPG at 20℃,the total LOX activities of N6-B,N6-M and N6-K reach 21.6,28.1 and 7.1 U/mL respectively.The yield of LOX in N6-B and N6-M is increased by 83%and 138%in contrast to N6(11.8 U/mL),respectively.The optimized induction by using orthogonal test is as follow:OD600=2.5,IPTG=2 mmol/mL,and inducing temperature is 20℃.The results show that the co-expression of superoxide dismutases could effectively improve the expression of LOX in E.coli,which provides a new idea for efficient heterologous expression of this enzyme.
作者
邱芳芳
刘松
堵国成
陈坚
QIU Fangfang;LIU Song;DU Guocheng;CHEN Jian(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2019年第10期23-29,共7页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目(31401638)
江苏省重点研发计划社会发展项目(BE2016629)
