Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli

Microbial Cell Factories

Research

Elisabeth Kurze¹  Ren Wei¹  Thorsten Oeser¹  Wolfgang Zimmermann¹  Christian Sonnendecker¹  Jinpeng Wang² 

[1] Institute of Biochemistry, Department of Microbiology and Bioprocess Technology, Leipzig University, Johannisallee 23, 04103, Leipzig, Germany;Institute of Biochemistry, Department of Microbiology and Bioprocess Technology, Leipzig University, Johannisallee 23, 04103, Leipzig, Germany;State Key Laboratory of Food Science and Technology, Jiangnan University, 214122, Wuxi, Jiangsu, China;

关键词: Bacillus;    Escherichia coli;    Cyclodextrin glucanotransferase;    Enzyme production;    Heterologous expression;    Signal peptides;    Protein secretion;   

DOI  :  10.1186/s12934-017-0701-1

 received in 2016-12-30, accepted in 2017-05-12,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundCyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed.ResultsExpression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg.ConclusionsThe signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme.

【 授权许可】

CC BY   
© The Author(s) 2017