Skip to main content
SpringerLink
Log in

Enhancement of Thermostability and Kinetic Efficiency of Aspergillus niger PhyA Phytase by Site-Directed Mutagenesis

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P < 0.05) with 24 and 22.6 % greater retention, respectively, compared with the PhyA of the wild type at 80 °C. The K m value of the improved thermostable P9 and P12 mutant enzymes for sodium phytate were 35 and 20 % lower (P < 0.05) with respect to the wild-type enzyme. In conclusion, it is feasible to simultaneously improve the thermostability and the catalytic efficiency of phytase to be used as an animal feed supplement.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Notes

  1. Thermostability: thermal stability and the maintenance of the enzyme activity at higher temperatures

References

  1. Viader-Salvado, J. M., et al. (2010). Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris. Applied and Environmental Microbiology, 76(19), 6423–30.

    Article  CAS  Google Scholar 

  2. Lehmann, M., et al. (2000). Exchanging the active site between phytases for altering the functional properties of the enzyme. Protein Science, 9(10), 1866–72.

    Article  CAS  Google Scholar 

  3. Kim, T., et al. (2006). Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive. Applied and Environmental Microbiology, 72(6), 4397–403.

    Article  CAS  Google Scholar 

  4. Abelson, P. H. (1999). A potential phosphate crisis. Science, 283(5410), 2015.

    Article  CAS  Google Scholar 

  5. Zhang, W., Mullaney, E. J., & Lei, X. G. (2007). Adopting selected hydrogen bonding and ionic interactions from Aspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase. Applied and Environmental Microbiology, 73(9), 3069–76.

    Article  CAS  Google Scholar 

  6. Mullaney, E. J., Daly, C. B., & Ullah, A. H. (2000). Advances in phytase research. Advances in Applied Microbiology, 47, 157–99.

    Article  CAS  Google Scholar 

  7. Lei, X. G., & Stahl, C. H. (2001). Biotechnological development of effective phytases for mineral nutrition and environmental protection. Applied Microbiology and Biotechnology, 57(4), 474–81.

    Article  CAS  Google Scholar 

  8. Liao, Y., Li, C. M., Chen, H., Wu, Q., Shan, Z., & Han, X. Y. (2013). Site-directed mutagenesis improves the thermostability and catalytic efficiency of Aspergillus niger N25 phytase mutated by I44E and T252R. Applied Biochemistry and Biotechnology, 171(4), 900–15.

    Article  CAS  Google Scholar 

  9. Lehmann, M., et al. (2000). From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Engineering, 13(1), 49–57.

    Article  CAS  Google Scholar 

  10. Zhang, W., & Lei, X. G. (2008). Cumulative improvements of thermostability and pH-activity profile of Aspergillus niger PhyA phytase by site-directed mutagenesis. Applied Microbiology and Biotechnology, 77(5), 1033–40.

    Article  CAS  Google Scholar 

  11. Oakley, A. J. (2010). The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic. Biochemical and Biophysical Research Communications, 397(4), 745–9.

    Article  CAS  Google Scholar 

  12. Liu, Q., et al. (2004). Crystallographic snapshots of Aspergillus fumigatus phytase, revealing its enzymatic dynamics. Structure, 12(9), 1575–83.

    Article  CAS  Google Scholar 

  13. Xiang, T., et al. (2004). Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine. Journal of Molecular Biology, 339(2), 437–45.

    Article  CAS  Google Scholar 

  14. Ma, Z.-Y., et al. (2011). A novel thermostable phytase from the fungus Aspergillus aculeatus RCEF 4894: gene cloning and expression in Pichia pastoris. World Journal of Microbiology and Biotechnology, 27(3), 679–686.

    Article  CAS  Google Scholar 

  15. Rodriguez, E., Mullaney, E. J., & Lei, X. G. (2000). Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme. Biochemical and Biophysical Research Communications, 268(2), 373–8.

    Article  CAS  Google Scholar 

  16. Hesampour A., O.R.S., M.A. Malboobi, J. Harati, N. Mohandesi (2014) Comparison of biochemical properties of recombinant phytase expression in favourable methylotrophic platforms, Pichia pastoris and Hansenula polymorpha. Progress in Biological Sciences, 4(1):95–109.

  17. Wang, X. Y., Meng, F. G., & Zhou, H. M. (2004). The role of disulfide bonds in the conformational stability and catalytic activity of phytase. Biochemistry and Cell Biology, 82(2), 329–34.

    Article  CAS  Google Scholar 

  18. Ullah, A. H. J., et al. (2012). A single mutation in the hepta-peptide active site of Aspergillus niger PhyA phytase leads to myriad biochemical changes. Advances in Microbiology, 2, 388–394.

    Article  CAS  Google Scholar 

  19. Liao, Y., Zeng, M., Wu, Z. F., Chen, H., Wang, H. N., Wu, Q., Shan, Z., & Han, X. Y. (2011). Improving phytase enzyme activity in a recombinant phyA mutant phytase from Aspergillus niger N25 by error-prone PCR. Applied Biochemistry and Biotechnology, 166(3), 549–62.

    Article  Google Scholar 

  20. Yao, M.Z., et al. (2012) Phytases: crystal structures, protein engineering and potential biotechnological applications. Journal of Applied Microbiology, 112(1):1–14.

  21. Eswar, N., et al. (2006) Comparative protein structure modeling using Modeller. Current Protocols in Protein Science, chapter 5:unit 5.6.

  22. Gunsteren, van W.F., S. R. Billeter, et al. (1996) Biomolecular simulations: the GROMOS96 manual and user guide. Zürich: VdF Hochschulverlag ETHZ.

  23. Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26, 283–291.

    Article  CAS  Google Scholar 

  24. Kabsch, W., & Sander, C. (1983). Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers, 22(12), 2577–637.

    Article  CAS  Google Scholar 

  25. Pronk, S., et al. (2013) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics, 29(7):845–54.

  26. Berendsen, H., Grigera, J., & Straatsma, T. (1987). The missing term in effective pair potentials. Journal of Physical Chemistry, 191, 6269–6271.

    Article  Google Scholar 

  27. Gunsteren, van W.F., Billeter S., Eising A., Hünenberger P.H., Krüger P., Mark A.E., Scott W., Tironi I.G (1996) Biomolecular simulation: the GROMOS96 manual and user guide. Zürich: VdF Hochschulverlag ETHZ.

  28. Bussi, G., Donadio, D., & Parrinello, M. (2007). Canonical sampling through velocity rescaling. Journal of Chemical Physics, 126(1), 014101.

    Article  Google Scholar 

  29. Parrinello, M., & Rahman, A. (1981). Polymorphic transitions in single crystals: a new molecular dynamics method. Journal of Applied Physics, 52, 7182–7190.

    Article  CAS  Google Scholar 

  30. Darden, T., York, D., & Pedersen, L. (1993). Particle mesh Ewald: an N-log (N) method for Ewald sums in large systems. Journal of Chemical Physics, 98(12), 10089–10092.

    Article  CAS  Google Scholar 

  31. Essmann, U., Perera L., Berkowitz M.L., Darden T., Lee H., Pedersen L.G. (1995) A smooth particle mesh Ewald method. Journal of Chemical Physics, 103:8577–8593.

  32. Bekker H., B.H.J.C., Fraaije J.G.E.M (1997) LINCS: a linear constraint solver for molecular simulations. Journal of Computational Chemistry, 18(12):1463–1472.

  33. Oakley, A.J. (2010) The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic. Biochemical and Biophysical Research Communication, 397(4):745–9.

  34. Zhong-You Ma, S.-C.P., Jing-Jing Jiang, Bo Huang, Mei-Zhen Fan, Zeng-Zhi Li (2011) A novel thermostable phytase from the fungus Aspergillus aculeatus RCEF 4894: gene cloning and expression in Pichia pastoris. World Journal of Microbiology and Biotechnology, 27(3):679–686.

  35. Ben Ali, M., et al. (2006). Thermostability enhancement and change in starch hydrolysis profile of the maltohexaose-forming amylase of Bacillus stearothermophilus US100 strain. Biochemistry Journal, 394(Pt 1), 51–6.

    CAS  Google Scholar 

  36. Santarossa, G., et al. (2005). Mutations in the “lid” region affect chain length specificity and thermostability of a Pseudomonas fragi lipase. FEBS Letters, 579(11), 2383–6.

    Article  CAS  Google Scholar 

  37. Lehmann, M., et al. (2002). The consensus concept for thermostability engineering of proteins: further proof of concept. Protein Engineering, 15(5), 403–11.

    Article  CAS  Google Scholar 

  38. Xiaoying, X., Shaoyang, J., Qimeng, M., & Tao, H. (2014). Heat treatment increases the bioactivity of C-terminally PEGylated staphylokinase. Process Biochemistry, 49, 1092–1096.

    Article  Google Scholar 

  39. Hushan, S. W. G., & Janitha, P. D. W. (2012). Molecular modelling for investigating structure–function relationships of soy glycinin. Trends in Food Science & Technology, 28, 153–167.

    Article  Google Scholar 

  40. Porto, C., Ferrara, M. C., Meli, M., Acampora, E., Avolio, V., & Rosa, M. (2012). Pharmacological enhancement of α-glucosidase by the allosteric chaperone N-acetylcysteine. Molecular Therapy, 20, 2201–2211.

    Article  CAS  Google Scholar 

  41. Rakesh, K., Ranvir, S., & Jagdeep, K. (2013). Characterization and molecular modelling of an engineered organic solvent tolerant, thermostable lipase with enhanced enzyme activity. Journal of Molecular Catalysis B: Enzymatic, 97, 243–251.

    Article  Google Scholar 

Download references

Acknowledgments

Support for this work by the Shahid Beheshti University and National Institute of Genetic Engineering and Biotechnology is greatly appreciated. We express our deep gratitude to Dr. H. Mollasalehi for his stylistic suggestions in the preparation of the manuscript and his expert editing.

Author information

Authors and Affiliations

  1. Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, Islamic Republic of Iran

    Ardeshir Hesampour

  2. Protein Research Centre, Shahid Beheshti University, Tehran, GC, Islamic Republic of Iran, P.O.Box: 19839-4716

    Seyed Ehsan Ranaei Siadat

  3. Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Islamic Republic of Iran

    Mohammad Ali Malboobi & Nooshin Mohandesi

  4. Department of Biophysics, School of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Seyed Shahriar Arab

  5. Department of Bioinformatics, School of Biological Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran

    Mohammad Mehdi Ghahremanpour

Authors
  1. Ardeshir Hesampour
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seyed Ehsan Ranaei Siadat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohammad Ali Malboobi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nooshin Mohandesi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Seyed Shahriar Arab
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mohammad Mehdi Ghahremanpour
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ardeshir Hesampour or Seyed Ehsan Ranaei Siadat.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hesampour, A., Siadat, S.E.R., Malboobi, M.A. et al. Enhancement of Thermostability and Kinetic Efficiency of Aspergillus niger PhyA Phytase by Site-Directed Mutagenesis. Appl Biochem Biotechnol 175, 2528–2541 (2015). https://doi.org/10.1007/s12010-014-1440-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-014-1440-y

Keywords

Search

Navigation