食品科学 ›› 2013, Vol. 34 ›› Issue (9): 143-149.doi: 10.7506/spkx1002-6630-201309030

• 生物工程 • 上一篇    下一篇

大肠杆菌重组表达磷脂酶C的发酵工艺优化

赵金星,张 梁*,顾正华,丁重阳,石贵阳   

  1. 江南大学 食品科学与技术国家重点实验室,工业生物技术教育部重点实验室,
    粮食发酵工艺与技术国家工程实验室,江苏 无锡 214122
  • 收稿日期:2013-04-02 修回日期:2013-04-22 出版日期:2013-05-15 发布日期:2013-05-07
  • 通讯作者: 张梁 E-mail:13861707271@139.com
  • 基金资助:

    国家“863”计划项目(2011AA100905);教育部“新世纪优秀人才支持计划”项目(NCET-11-0665);
    江南大学食品科学与技术国家重点实验室自由探索资助课题(SKLF-ZZA-201201)

Optimization of Fermentation Process for Recombinant Escherichia coli Expression of Phospholipase C

ZHAO Jin-xing,ZHANG Liang*,GU Zheng-hua,DING Zhong-yang,SHI Gui-yang   

  1. State Key Laboratory of Food Science and Technology, Key Laboratory of Industrial Biotechnology, Ministry of Education, National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China
  • Received:2013-04-02 Revised:2013-04-22 Online:2013-05-15 Published:2013-05-07
  • Contact: ZHANG Liang E-mail:13861707271@139.com

摘要:

以重组表达铜绿假单胞菌Pseudomonas aeruginosa 41磷脂酶C的大肠杆菌BL21 (DE3)/pET28a-plcH为考察菌株,从TB培养基出发,在优化缓冲液、碳源种类和质量浓度的基础上,比较异丙基-β-D-硫代吡喃半乳糖苷(IPTG)、乳糖诱导重组酶表达的效果,并考察甘氨酸对重组酶发酵的影响;最后,在7L发酵罐规模对上述优化工艺进行放大验证。结果表明:重组大肠杆菌最适培养基组成为:蛋白胨12g/L、酵母膏24g/L、甘油5g/L,0.25mol/L Tris-HCl(pH7.2~7.4)缓冲液配制;在此培养基中添加卡那霉素(Kana)的质量浓度至0.15mg/mL,37℃、200r/min培养6h后,添加乳糖至终质量浓度为5g/L,在25℃、150r/min诱导培养20 h,重组磷脂酶C活力可达到(1422.42±37.17)U/mL;7L发酵罐实验中,总酶活力达到(11583.35±70.21)U/mL,比摇瓶条件下提高了7倍左右,其中胞内酶活力最高为(10957.97±58.03)U/mL,胞外酶活力最高为(645.27±13.87)U/mL。

关键词: 磷脂酶C, 重组大肠杆菌, 发酵过程, 工艺优化

Abstract:

The culture condition of Escherichia coli BL21 (DE3)/pET28a-plcH expressing the phospholipase C gene from
Pseudomonas aeruginosa 41 was optimized. The expression vector was cultured in TB medium and important parameters
such as the type of bufffer and the type and concentration of nitrogen source were optimized. Furthermore, the inducive
influences of IPTG, lactose and glycine on phospholipase C production were comparatively discussed. The optimized
medium composition was determined as (g/L): trytone 12, yeast extrat 24, glycerol 5, and Tris-HCl (0.25 mol/L, pH 7.2—7.4).
After sterilization, 0.15 g/L kanamycin was supplemented. In conical flasks, strain E. coli BL21 (DE3)/pET28a-plcH was
incubated at 37 ℃ and 200 r/min for six hours, followed by addition of 5 g/L lactose, and then the culture was maintained
at 25 ℃ and 150 r/min for twenty more hours. Under the optimized conditions, the final activity of phospholipase C was
(1422.42±37.17) U/mL. In a 7 L fermentor, the total activity reached (11583.35±70.21) U/mL with an intracellular activity of
(10957.97±58.03) U/mL and an extracellular activity of (645.27±13.87) U/mL.

Key words: phospholipase C, recombinant E. coli, fermentation process, optimization

中图分类号: