生物技术通报 ›› 2021, Vol. 37 ›› Issue (2): 138-148.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0529

• 研究报告 • 上一篇    下一篇

南极假丝酵母脂肪酶B基因在大肠杆菌中的表达和发酵优化

吴蓉1(), 曹佳睿1, 曹君1, 刘飞翔1, 杨猛1, 苏二正1,2()   

  1. 1.南京林业大学轻工与食品学院,南京 210037
    2.南京林业大学南方现代林业协同创新中心 南京林业大学林业资源高效加工利用协同创新中心,南京 210037
  • 收稿日期:2020-05-25 出版日期:2021-02-26 发布日期:2021-02-26
  • 作者简介:吴蓉,女,博士研究生,研究方向:酶与生物催化;E-mail: rongwu@njfu.edu.cn
  • 基金资助:
    江苏省“六大人才高峰”高层次人才项目(2015-JY-016);中国博士后科学基金(2016M600417);中国博士后科学基金(2017T100373);江苏省第五期“333 工程”培养资金资助项目(BRA2017458)

Expression and Fermentation Optimization of Candida antarctica Lipase B in Escherichia coli

WU Rong1(), CAO Jia-rui1, CAO Jun1, LIU Fei-xiang1, YANG Meng1, SU Er-zheng1,2()   

  1. 1. College of Light Industry and Food Engineering,Nanjing Forestry University,Nanjing 210037
    2. Co-innovation Center for the Sustainable Forestry in Southern China,Nanjing Forestry University,Co-Innovation Center for Efficient Processing and Utilization of Forest Products,Nanjing Forestry University,Nanjing 210037
  • Received:2020-05-25 Published:2021-02-26 Online:2021-02-26

摘要:

旨在利用大肠杆菌实现南极假丝酵母脂肪酶B(CALB)基因的高效可溶性表达,并降低生产成本。构建带有不同信号肽的CALB基因表达质粒,转化至不同大肠杆菌宿主中,在摇瓶中进行基础培养基、诱导条件、培养基组成成分和进程曲线的优化。结果显示,带有PelB信号肽的重组菌pET25b-CALB-1/Rosetta(DE3)在20℃下使用0.5%(W/V)乳糖在TY培养基中诱导表达效果最好,优化后的合成培养基成分为1.75%(W/V)山梨醇、2.25%(W/V)鱼蛋白胨、1%(W/V)安琪酵母提取物、0.75%(W/V)Na2HPO4。在摇瓶中诱导60 h后,CALB酶活力最高达到35.67 U/mL,相比于初始发酵酶活力提高了17.77倍,是目前大肠杆菌生产CALB的最高水平。成功地构建了大肠杆菌表达系统,经过系统优化后,CALB的高水平可溶性表达得以实现。

关键词: 南极假丝酵母脂肪酶B, 大肠杆菌, 可溶性表达, 发酵优化

Abstract:

In order to achieve efficient soluble expression of Candida antarctica lipase B(CALB)in Escherichia coli at lower cost,CALB gene expression plasmids with different signal peptides were constructed and transformed into different E. coli hosts,and then a series of fermentation experiments were employed in shaking flasks to optimize the culture medium types,inducing conditions,components of medium and time course of cultivation. The results showed that the recombinant strain pET25b-CALB-1/Rosetta(DE3)with PelB signal peptide was induced to have the best expression in TY medium at 20℃ employing 0.5%(W/V)lactose. The optimal composition of fermentation medium was 1.75%(W/V)sorbitol,2.25%(W/V)fish peptone,1%(W/V)Angel yeast extract and 0.75%(W/V)Na2HPO4. The recombination system achieved a maximum CALB activity of 35.67 U/mL after induction for 60 h,which was 17.77 times higher than that before optimization,i.e.,the highest one of CALB produced by E. coli at present. In conclusion,the expression system in E. coli was successfully established,and the high-level soluble expression of CALB was obtained after system optimization.

Key words: Candida antarctica lipase B, Escherichia coli, soluble expression, fermentation optimization