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›› 2008, Vol. 8 ›› Issue (1): 140-143.

• 生化工程专栏 • 上一篇    下一篇

产GL-7ACA酰化酶基因工程菌的高活性表达

林晨露,李强,刘亚飞,王波   

  1. 清华大学化学工程系
  • 出版日期:2008-02-20 发布日期:2008-02-20

Over-expression of GL-7-ACA Acylase Gene in E. coli

LIN Chen-lu,LI Qiang,LIU Ya-fei,WANG Bo   

  1. Department of Chemical Engineering , Tsinghua University
  • Online:2008-02-20 Published:2008-02-20

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摘要： 实现了戊二酰基-7-氨基头孢烷酸(GL-7ACA)酰化酶的组成型表达，并通过替换重组质粒启动子的RBS序列，在摇瓶培养条件下，使新构建的重组质粒pGEMKT-HPRfACY在大肠杆菌JM105中表达GL-7ACA酰化酶酶活达到0.44 U/mL，是替换前的2倍. 使用廉价的玉米浆作为培养基中氮源主要来源，使JM105/pGEMKT-HPRfACY菌株酶活提高到2.98 U/mL. 采用补料批式培养，利用流加营养物控制发酵中后期的pH值，在pH为7.5的条件下，酶活峰值达到6.37 U/mL. 该体系无需诱导，工艺操作过程简单，成本低廉.

关键词: 7-氨基头孢烷酸, 戊二酰基-7-氨基头孢烷酸酰化酶, 高密度发酵

Abstract: The constitutive expression of glutaryl-7-amidocephalosporanic acid (GL-7-ACA) acylase was realized. Based on it, a new plasmid pGEMKT-HPfACY with optimized RBS was constructed and transformed into E. coli JM105. After culturing in LB media, this recombinant strain reached an activity of 0.44 U/mL. This activity was twice of former one while the activity of the strain JM105/pGEMKT-HPRfACY reached as high as 2.98 U/mL using corn-steep as culture media. The acylase activity of recombinant strain hit 6.37 U/mL, using a strategy of controlling pH 7.5 by adding nutrient such as glycerin.

Key words: 7-aminocephalosporanic acid, glutaryl-7-amidocephalosporanic acid acylase, constitutive expression