Advertisement

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

  • Sirima Mingmongkolchai
  • Watanalai Panbangred
Article

Abstract

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.

Keywords

Bacillus subtilis CotG Phytase Spores Spore surface display Peptide linker 

Notes

Acknowledgments

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)

References

  1. 1.
    Kumar, V., Singh, D., Sangwan, P., & Gill, P. K. (2015). Management of environmental phosphorus pollution using phytases: current challenges and future prospects. In Applied environmental biotechnology: present scenario and future trends (pp. 97–114). Berlin: Springer.Google Scholar
  2. 2.
    Gupta, R. K., Gangoliva, S. S., & Singh, N. K. (2015). Reduction of phytic acid and enhancement of bioavailable micronutrients in food grains. Journal of Food Science and Technology, 52(2), 676–684.CrossRefGoogle Scholar
  3. 3.
    Dersjant-Li, Y., Awati, A., Schulze, H., & Partridge, G. (2015). Phytase in non-ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. Journal of the Science of Food and Agriculture, 95(5), 878–896.CrossRefGoogle Scholar
  4. 4.
    Selle, P. H., & Ravindran, V. (2008). Phytate-degrading enzymes in pig nutrition. Livestock Science, 113(2–3), 99–122.CrossRefGoogle Scholar
  5. 5.
    Igbasan, F. A., Männer, K., Miksch, G., Borriss, R., Farouk, A., & Simon, O. (2000). Comparative studies on the in vitro properties of phytases from various microbial origins. Archiv fur Tierernahrung, 53(4), 353–373.CrossRefGoogle Scholar
  6. 6.
    Morales, G. A., Moyano, F. J., & Marquez, L. (2011). In vitro assessment of the effects of phytate and phytase on nitrogen and phosphorus bioaccessibility within fish digestive tract. Animal Feed Science Technology, 170(3–4), 209–221.CrossRefGoogle Scholar
  7. 7.
    Rutherford, N., & Mourez, M. (2006). Surface display of proteins by Gram-negative bacterial autotransporters. Microbial Cell Factories, 5(1), 22.CrossRefGoogle Scholar
  8. 8.
    Wang, G., Xia, Y., Gu, Z., Zhang, H., Chen, Y. Q., Chen, H., Ai, L., & Chen, W. (2015). A new potential secretion pathway for recombinant proteins in Bacillus subtilis. Microbial Cell Factories, 14(1), 179.CrossRefGoogle Scholar
  9. 9.
    Chen, H., Ullah, J., & Jia, J. (2017). Progress in Bacillus subtilis spore surface display technology towards environment, vaccine development, and biocatalysis. Journal of Molecular Microbiology and Biotechnology, 27(3), 159–167.CrossRefGoogle Scholar
  10. 10.
    Wong, S. L. (1995). Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Current Opinion in Biotechnology, 6(5), 517–522.CrossRefGoogle Scholar
  11. 11.
    Kim, J., & Schumann, W. (2009). Display of proteins on Bacillus subtilis endospores. Cellular and Molecular Life Sciences, 66(19), 3127–3136.CrossRefGoogle Scholar
  12. 12.
    Henrigues, A. O., & Moran, C. P. (2007). Structure, assembly, and function of the spore surface layers. Annual Review of Microbiology, 61(1), 555–588.CrossRefGoogle Scholar
  13. 13.
    McKenney, P. T., Driks, A., & Eichenberger, P. (2013). The Bacillus subtilis endospores: assembly and functions of the multilayered coat. Nature Reviews Microbiology, 11(1), 33–44.CrossRefGoogle Scholar
  14. 14.
    Chen, H., Tian, R., Ni, Z., Zhang, T., Chen, Z., Chen, K., & Yang, S. (2015). Surface display of the thermophilic lipase Tm1350 on the spore of Bacillus subtilis. Extremophiles, 19(4), 799–808.CrossRefGoogle Scholar
  15. 15.
    Isticato, R., Cangiano, G., Tran, H. T., Ciabattini, A., Medaglini, D., Oggioni, M. R., De Felice, M., Pozzi, G., & Ricca, E. (2001). Surface display of recombinant proteins on Bacillus subtilis spores. Journal of Bacteriology, 183(21), 6294–6301.CrossRefGoogle Scholar
  16. 16.
    Mauriello, E. M., Duc le, H., Isticato, R., Cangiano, G., Hong, H. A., De Felice, M., Ricca, E., & Cutting, S. M. (2004). Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine, 22(9), 1177–1187.CrossRefGoogle Scholar
  17. 17.
    Wang, N., Chang, C., Yao, Q., Li, G., Qin, L., Chen, L., & Chen, K. (2011). Display of Bombyx mori alcohol dehydrogenases on the Bacillus subtilis spore surface to enhance enzymatic activity under adverse conditions. PLoS One, 6(6), e21454.CrossRefGoogle Scholar
  18. 18.
    Hwang, B. Y., Kim, B. G., & Kim, J. H. (2011). Bacterial surface display of a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis using the Bacillus subtilis spore display system. Bioscience Biotechnology and Biochemistry, 75(9), 1862–1865.CrossRefGoogle Scholar
  19. 19.
    Chen, H., Chen, Z., Ni, Z., Tian, R., Zhang, T., Jia, J., Chen, K., & Yang, S. (2016). Display of Thermotoga maritima MSB8 nitrilase on the spore surface of Bacillus subtilis using out coat protein CotG as the fusion partner. Journal of Molecular Catalysis B: Enzymatic, 123, 73–80.CrossRefGoogle Scholar
  20. 20.
    Potot, S., Serra, C. R., Henriques, A. O., & Schyns, G. (2010). Display of recombinant proteins on Bacillus subtilis spores, using a coat-associated enzyme as the carrier. Applied and Environmental Microbiology, 76(17), 5926–5933.CrossRefGoogle Scholar
  21. 21.
    Wang, H., Yang, R., Hua, X., Zhao, W., & Zhang, W. (2015). Functional display of active β-galactosidase on Bacillus subtilis spores using crust proteins as carriers. Food Science and Biotechnology, 24(5), 1755–1759.CrossRefGoogle Scholar
  22. 22.
    Hosseini-Abari, A., Kim, B. G., Lee, S. H., Emtiazi, G., Kim, W., & Kim, J. H. (2016). Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein. Journal of Basic Microbiology, 56(12), 1331–1337.CrossRefGoogle Scholar
  23. 23.
    Panbangred, W., Fukusaki, E., Epifanio, E. C., Shinmyo, A., & Okada, H. (1985). Expression of a xylanase gene of Bacillus pumilus in Escherichia coli and Bacillus subtilis. Applied Microbiology and Biotechnology, 22(4), 259–264.CrossRefGoogle Scholar
  24. 24.
    Cho, E. A., Seo, J., Lee, D. W., & Pan, J. G. (2011). Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores. Enzyme and Microbial Technology, 49(1), 100–104.CrossRefGoogle Scholar
  25. 25.
    Sánchez, B., Arias, S., Chaignepain, S., Denayrolles, M., Schmitter, J. M., Bressollier, P., & Urdaci, M. C. (2009). Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin. Microbiology, 155(5), 1708–1716.CrossRefGoogle Scholar
  26. 26.
    Shields, D. C., & Sharp, P. M. (1987). Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases. Nucleic Acids Research, 15(19), 8023–8040.CrossRefGoogle Scholar
  27. 27.
    Hinc, K., Iwanicki, A., & Obuchowski, M. (2013). New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microbial Cell Factories, 12(1), 22.CrossRefGoogle Scholar
  28. 28.
    Lu, Y.-P., Zhang, C., Lv, F. X., Bie, X. M., & Lu, Z.-X. (2012). Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis. Letters in Applied Microbiology, 55(1), 9–14.CrossRefGoogle Scholar
  29. 29.
    Monroe, A., & Setlow, P. (2006). Localization of the transglutaminase cross-linking sites in the Bacillus subtilis spore coat protein GerQ. Journal of Bacteriology., 188(21), 7609–7616.CrossRefGoogle Scholar
  30. 30.
    Lei, X. G., & Porres, J. M. (2003). Phytase enzymology, applications, and biotechnology. Biotechnology Letters, 25(21), 1787–1794.CrossRefGoogle Scholar
  31. 31.
    Miksch, G., Kleist, S., Friehs, K., & Flaschel, E. (2002). Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors. Applied Microbiology and Biotechnology, 59(6), 685–694.CrossRefGoogle Scholar
  32. 32.
    Tai, H.-M., Yin, L.-J., Chen, W.-C., & Jiang, S.-T. (2013). Overexpression of Escherichia coli phytase in Pichia pastoris and its biochemical properties. Journal of Agricultural and Food Chemistry, 61(25), 6007–6015.CrossRefGoogle Scholar
  33. 33.
    Lee, S., Kim, T., Stahl, C. H., & Lei, X. G. (2005). Expression of Escherichia coli AppA2 phytase in four yeast systems. Biotechnology Letters, 27(5), 327–334.CrossRefGoogle Scholar
  34. 34.
    Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5, 172.Google Scholar
  35. 35.
    Buckholz, R. G. (1993). Yeast systems for the expression of heterologous gene products. Current Opinion in Biotechnology, 4(5), 538–542.CrossRefGoogle Scholar
  36. 36.
    Golovan, S., Wang, G., Zhang, J., & Forsberg, C. W. (2000). Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Canadian Journal of Microbiology, 46(1), 59–71.CrossRefGoogle Scholar
  37. 37.
    Greiner, R., Konietzny, U., & Jany, K. D. (1993). Purification and characterization of two phytases from Escherichia coli. Archives Biochemistry Biophysics, 303(1), 107–113.CrossRefGoogle Scholar
  38. 38.
    Simon, O., & Igbasan, F. (2002). In vitro properties of phytases from various microbial origins. International Journal of Food Science and Technology, 37(7), 813–822.CrossRefGoogle Scholar
  39. 39.
    Kiarie, E., Woyengo, T., & Nyachoti, C. M. (2015). Efficacy of new 6-phytase from Buttiauxella spp. on growth performance and nutrient retention in broiler chickens fed corn soybean meal-based diets. Asian-Australasian Journal of Animal Sciences, 28(10), 1479–1487.CrossRefGoogle Scholar
  40. 40.
    Menezes-Blackburn, D., Jorquera, M., Gianfreda, L., Rao, M., Greiner, R., Garrido, E., & de la Luz Mora, M. (2011). Activity stabilization of Aspergillus niger and Escherichia coli phytases immobilized on allophanic synthetic compounds and montmorillonite nanoclays. Bioresource Technology, 102(20), 9360–9367.CrossRefGoogle Scholar
  41. 41.
    Cho, E. A., Kim, E. J., & Pan, J. G. (2011). Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores. Enzyme and Microbial Technology, 49(1), 66–71.CrossRefGoogle Scholar
  42. 42.
    Ullah, J., Chen, H., Vastermark, A., Jia, J., Wu, B., Ni, Z., Le, Y., & Wang, H. (2017). Impact of orientation and flexibility of peptide linkers on T. maritima lipase Tm1350 displayed on Bacillus subtilis spores surface using CotB as fusion partner. World Journal of Microbiology and Biotechnology, 33(9), 166.CrossRefGoogle Scholar
  43. 43.
    Chen, H., Wu, B., Zhang, T., Jia, J., Lu, J., Chen, Z., Ni, Z., & Tan, T. (2017). Effect of linker length and flexibility on the Clostridium thermocellum esterase displayed on Bacillus subtilis spores. Applied Biochemistry and Biotechnology, 182(1), 168–180.CrossRefGoogle Scholar

Copyright information

© 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

Personalised recommendations