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›› 2007, Vol. 7 ›› Issue (6): 1187-1191.

• 生化工程专栏 • 上一篇    下一篇

argE基因的表达优化及Zn2+添加时间和浓度的影响机制分析

李环,陈悦,翁秋萍,朱大伟,韦萍   

  1. 南京工业大学制药与生命科学学院
  • 出版日期:2007-12-20 发布日期:2007-12-20

Expressing Optimization of argE Gene and Mechanism Analysis of Zn2+ Feeding Time and Its Concentration

LI Huan,CHEN Yue,WENG Qiu-ping,ZHU Da-wei,WEI Ping   

  1. College of Life Science and Pharmacy, Nanjing University of Technology
  • Online:2007-12-20 Published:2007-12-20

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摘要： 确定了目的基因argE在重组菌BL21(DE3)-pET22b-argE中的表达位置，研究了Zn2+对重组菌生长及表达产物活性的影响，并分析了影响机制. 结果表明，argE可在重组菌中高效表达，表达产物N-乙酰鸟氨酸脱酰基酶大多以不可溶的包涵体形式存在，只有少量为有活性的可溶性表达. 1.0 g/L的Mg2+对重组菌的生长及酶活有明显促进作用. Zn2+加入时机及加入量不同，影响结果也不同. 发酵起始加入Zn2+严重抑制菌的生长及酶活，而在1.0%乳糖诱导2.5 h后加入则可解除生长抑制并提高酶活. SDS-PAGE电泳及活力测定证实Zn2+参与形成酶的催化中心，对酶的表达量没有影响.

关键词: argE基因, 表达优化, Zn2+加入时机, 影响机制, 活性表达

Abstract: The cellular location of target gene argE expressed in recombinant BL21(DE3)-pET22b-argE was determined. The effects of Zn2+ effect feeding time and concentration on the biomass production and gene expression product activity were studied. The mechanism of Zn2+ effect was analyzed. The results showed that N-acetylornithine deacetylase coded by argE gene was over expressed and most of it was expressed as insoluble inclusion body, only a little as soluble expression. 1.0 g/L Mg2+ concentration improved both the biomass production and enzyme activity greatly. With different concentrations and feeding times, Zn2+ could bring about different influences. In contrast to the inhibition of feeding Zn2+ at the cultivation beginning, addition of 1.0 mg/L Zn2+ after inducing for 2.5 h with 1.0% lactose reduced the inhibition on the growth and increased the enzyme activity. SDS-PAGE analysis and enzyme activity measurement proved that Zn2+ did not influence the expression rate, but the catalyst site of recombinant NAOase.

Key words: argE gene, expression optimization, feeding time of Zn2+, mechanism, activity expression