SCI和EI收录∣中国化工学会会刊

›› 2016, Vol. 24 ›› Issue (7): 909-913.DOI: 10.1016/j.cjche.2016.02.001

• Biotechnology and Bioengineering • 上一篇    下一篇

Permeabilization of Escherichia coli with ampicillin for a whole cell biocatalyst with enhanced glutamate decarboxylase activity

Weirui Zhao1,2, Sheng Hu1, Jun Huang3, Piyu Ke1,2, Shanjing Yao2, Yinlin Lei1, Lehe Mei1,2, Jinbo Wang1   

  1. 1 School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China;
    2 Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;
    3 School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
  • 收稿日期:2015-04-07 修回日期:2015-07-24 出版日期:2016-07-28 发布日期:2016-08-17
  • 通讯作者: Lehe Mei
  • 基金资助:
    Supported by the grants from the National Natural Science Foundation of China (21176220, 20876143, 31470793), the Natural Science Foundation of Zhejiang Province (Z13B060008) and the Key Technology Research and Development Project of Ningbo (2011C11023).

Permeabilization of Escherichia coli with ampicillin for a whole cell biocatalyst with enhanced glutamate decarboxylase activity

Weirui Zhao1,2, Sheng Hu1, Jun Huang3, Piyu Ke1,2, Shanjing Yao2, Yinlin Lei1, Lehe Mei1,2, Jinbo Wang1   

  1. 1 School of Biotechnology and Chemical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China;
    2 Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;
    3 School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
  • Received:2015-04-07 Revised:2015-07-24 Online:2016-07-28 Published:2016-08-17
  • Supported by:
    Supported by the grants from the National Natural Science Foundation of China (21176220, 20876143, 31470793), the Natural Science Foundation of Zhejiang Province (Z13B060008) and the Key Technology Research and Development Project of Ningbo (2011C11023).

摘要: The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability barrier against the diffusion of substrates and products. Although common chemical or physical permeabilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, aswell as impede large scale processing. To increase membrane permeability and cellbound glutamate decarboxylase (GAD) activity of recombinant Escherichia coli (BL21(DE3)-pET28a-gadB) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or surfactants to the culture media. Ampicillin was the most effective at improving cell-bound GAD activity of the BL21(DE3)-pET28a-gadB, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 μg·ml-1. Using this concentration, the cell biomass did decrease by 40.58%, but the cell-bound GAD activity of BL21(DE3)-pET28a-gadB and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment of BL21 (DE3)-pET28a-gadB cells with 5 μg·ml-1 ampicillin resulted in structural changes to the cell envelope, but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol (PVA), alginate, and boric acid, the transformation rate of γ-aminobutyric acid (GABA) at the 10th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after 30 days of storage at 4℃.

关键词: γ-Aminobutyric acid, Ampicillin, Escherichia. coli, Glutamate decarboxylase, Permeabilization

Abstract: The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability barrier against the diffusion of substrates and products. Although common chemical or physical permeabilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, aswell as impede large scale processing. To increase membrane permeability and cellbound glutamate decarboxylase (GAD) activity of recombinant Escherichia coli (BL21(DE3)-pET28a-gadB) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or surfactants to the culture media. Ampicillin was the most effective at improving cell-bound GAD activity of the BL21(DE3)-pET28a-gadB, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 μg·ml-1. Using this concentration, the cell biomass did decrease by 40.58%, but the cell-bound GAD activity of BL21(DE3)-pET28a-gadB and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment of BL21 (DE3)-pET28a-gadB cells with 5 μg·ml-1 ampicillin resulted in structural changes to the cell envelope, but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol (PVA), alginate, and boric acid, the transformation rate of γ-aminobutyric acid (GABA) at the 10th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after 30 days of storage at 4℃.

Key words: γ-Aminobutyric acid, Ampicillin, Escherichia. coli, Glutamate decarboxylase, Permeabilization