Display of Escherichia coli Phytase on the Surface of Bacillus subtilis Spore Using CotG as an Anchor Protein

  • Sirima Mingmongkolchai
  • Watanalai PanbangredEmail author


Escherichia coli phytase (AppA) has been widely used as an exogenous feed enzyme for monogastric animals; however, the production of this enzyme has been examined primarily in E. coli and yeast expression systems. As an alternative to production of soluble phytase, an enzyme immobilization method using the Bacillus subtilis spore outer-coat protein CotG as an anchoring motif for the display of the AppA was attempted. Using this motif, AppA was successfully produced on the spore surface of B. subtilis as verified by Western blot analysis and phytase activity measurements. Analysis of the pH stability indicated that more than 50% activity was retained after incubation at four different pH values (2.0, 4.0, 7.0, and 8.0) for up to 12 h, with maximum activity observed at pH 4.5. The highest enzyme activity seen at 55 °C and thermal stability measurements demonstrated that more than 30% activity remained after 30 min incubation at 60 °C. The spore surface-displayed AppA was resistant to pepsin, and more stable than phytase produced previously using a yeast expression system. Furthermore, we present data indicating that the use of peptide linkers may help improve the bioactivity of displayed enzymes on the spore surface of B. subtilis.


Bacillus subtilis CotG Phytase Spores Spore surface display Peptide linker 



We would like to thank Assoc. Prof. Dr. Laran T. Jensen for kindly proofreading this manuscript.

Funding Information

This study was supported by grants (PHD56I0013) from Research and Researcher for Industries (RRI), Thailand Research Fund (TRF) in collaboration with Betagro Public Company Limited.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12010_2018_2855_MOESM1_ESM.docx (848 kb)
ESM 1 (DOCX 848 kb)


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biotechnology, Faculty of ScienceMahidol UniversityBangkokThailand
  2. 2.Mahidol University-Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU:CRC), Faculty of ScienceMahidol UniversityBangkokThailand

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