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Soluble expression of pullulanase from Bacillus acidopullulyticus in Escherichia coli by tightly controlling basal expression

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Bacillus acidopullulyticus pullulanase (BaPul13A) is a widely used debranching enzyme in the starch industry. A few details have been reported on the heterologous expression of BaPul13A in Escherichia coli (E. coli). This study compares different E. coli expression systems to improve the soluble expression level of BaPul13A. When pET22b(+)/pET28a(+) was used as the expression vector, the soluble expression of BaPul13A can be achieved by tightly controlling basal expression, whereas pET-20b(+)/pGEX4T2 leads to insoluble inclusion bodies. An efficient process control strategy aimed at minimizing the formation of inclusion bodies and enhancing the production of pullulanase was developed by a step decrease of the temperature in a 5-L fermentor. The highest total enzyme activity of BaPul13A reached 1,156.32 U/mL. This work reveals that the T7 promoter with lac operator and lacI gene collectively contribute to the soluble expression of BaPul13A, whereas either a T7 promoter alone or combined with the lac operator and lacI gene results in poor solubility. Basal expression in the initial growth phase of the host significantly affects the solubility of BaPul13A in E. coli.

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Acknowledgments

The authors would like to thank Wenwen Guo and Lu Li for the drawing figures. This work was supported by the National Basic Research Program of China (973 Program) (Grant No.: 2013CB733602) and the Fundamental Research Funds for the Central Universities (Grant No. JUSRP51401A).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China

    Ana Chen, Yamei Li, Xiuxia Liu, Quan Long, Yankun Yang & Zhonghu Bai

  2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Ana Chen & Yamei Li

  3. School of Biochemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China

    Ana Chen

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  1. Ana Chen
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  2. Yamei Li
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Correspondence to Yankun Yang or Zhonghu Bai.

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Chen, A., Li, Y., Liu, X. et al. Soluble expression of pullulanase from Bacillus acidopullulyticus in Escherichia coli by tightly controlling basal expression. J Ind Microbiol Biotechnol 41, 1803–1810 (2014). https://doi.org/10.1007/s10295-014-1523-3

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  • DOI: https://doi.org/10.1007/s10295-014-1523-3

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