Skip to main content
SpringerLink
Log in

Recombinant HAP Phytase of the Thermophilic Mold Sporotrichum thermophile: Expression of the Codon-Optimized Phytase Gene in Pichia pastoris and Applications

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The codon-optimized phytase gene of the thermophilic mold Sporotrichum thermophile (St-Phy) was expressed in Pichia pastoris. The recombinant P. pastoris harboring the phytase gene (rSt-Phy) yielded a high titer of extracellular phytase (480 ± 23 U/mL) on induction with methanol. The recombinant phytase production was ~40-fold higher than that of the native fungal strain. The purified recombinant phytase (rSt-Phy) has the molecular mass of 70 kDa on SDS-PAGE, with K m and V max (calcium phytate), k cat and k cat/K m values of 0.147 mM and 183 nmol/mg s, 1.3 × 103/s and 8.84 × 106/M s, respectively. Mg2+ and Ba2+ display a slight stimulatory effect, while other cations tested exert inhibitory action on phytase. The enzyme is inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward’s reagent K and 2,3-bunatedione, but resistant to both pepsin and trypsin. The rSt-Phy is useful in the dephytinization of broiler feeds efficiently in simulated gut conditions of chick leading to the liberation of soluble inorganic phosphate with concomitant mitigation in antinutrient effects of phytates. The addition of vanadate makes it a potential candidate for generating haloperoxidase, which has several applications.

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

Similar content being viewed by others

References

  1. Akita, M., Kayatama, K., Hatada, Y., Ito, S., & Horikoshi, K. (2005). A novel β-glucanase gene from Bacillus halodurans C-125. FEMS Microbiology Letters, 248, 9–15.

    Article  CAS  Google Scholar 

  2. Arjmand, S., Lotfi, A. S., Shamsara, M., & Mowla, S. J. (2013). Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris. Electronic Journal of Biotechnology,. doi:10.2225/vol16-issue1-fulltext-4.

    Google Scholar 

  3. Bohn, L., Meyer, A. S., & Rasmussen, S. K. (2008). Phytate: Impact on environment and human nutrition a challenge for molecular breeding. Journal of Zheijang University Science B, 9, 165–191.

    Article  CAS  Google Scholar 

  4. Casey, A., & Walsh, G. (2004). Identification and characterization of a phytase of potential commercial interest. Journal of Biotechnology, 110, 313–322.

    Article  CAS  Google Scholar 

  5. Cereghino, G., Sunga, A., Cereghino, J. L., & Cregg, J. M. (2002). Genetic engineering. US: Springer.

    Google Scholar 

  6. Chauthaiwale, J., & Rao, M. (1994). Chemical modification of xylanase from alkalothermophilic Bacillus species: Evidence for essential carboxyl group. Biochimica et Biophysica Acta, 1204, 164–168.

    Article  CAS  Google Scholar 

  7. Cregg, J. M., Cereghino, J. L., Shi, J. Y., & Higgins, D. R. (2000). Recombinant protein expression in Pichia pastoris. Molecular Biotechnology, 16, 23–52.

    Article  CAS  Google Scholar 

  8. Delroisse, J. M., et al. (2005). Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris. Protein Expression and Purification, 42, 286–294.

    Article  CAS  Google Scholar 

  9. Dixon, M., & Webb, E. C. (1979). Enzyme kinetics. In M. Dixon & E. C. Webb (Eds.), Enzymes (pp. 47–206). New York: Academic Press.

    Google Scholar 

  10. Ehrlich, K. C., Montalbano, B. G., Mullaney, E. J., Dischinger, H. C., & Ullah, A. H. J. (1993). Identification and cloning of a second phytase gene (phyB) from Aspergillus niger. Biochemical and Biophysical Research Communications, 195, 53–57.

    Article  CAS  Google Scholar 

  11. Fonseca-Maldonado, R., Maller, A., Bonneil, E., Thibault, P., Botelho-Machado, C., Ward, R. J., & Polizeli Mde, L. (2014). Biochemical properties of glycosylation and characterization of a histidine acid phosphatase (phytase) expressed in Pichia pastoris. Protein Expression and Purification, 99, 43–49.

    Article  CAS  Google Scholar 

  12. Fu, D., Li, Z. Y., Huang, H. Q., Yuan, T., Shi, P., Luo, H., et al. (2011). Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site. Applied Microbiology and Biotechnology, 90, 1295–1302.

    Article  CAS  Google Scholar 

  13. Gulati, H. K., Chadha, B. S., & Saini, H. S. (2007). Production, purification and characterization of thermostable phytase from thermophilic fungus Thermomyces lanuginosus TL-7. Acta Microbiologica et Immunologica Hungarica, 54, 121–138.

    Article  CAS  Google Scholar 

  14. Gustafsson, C., Govindarajan, S., & Minshull, J. (2004). Codon bias and heterologous protein expression. Trends in Biotechnology, 22, 346–353.

    Article  CAS  Google Scholar 

  15. Hemrika, W., Renirie, R., Dekker, H. L., Barnett, P., & Wever, R. (1997). From phosphatases to vanadium peroxidases: A similar architecture of the active site. PNAS USA, 94, 2145–2149.

    Article  CAS  Google Scholar 

  16. Hunter-Cevera, J. C., & Sotos, L. (1986). Screening for a “new” enzyme in nature: Haloperoxidase production by Death Valley dematiaceous hyphomycetes. Microbial Ecology, 12, 121–127.

    Article  CAS  Google Scholar 

  17. Joshi, S., & Satyanarayana, T. (2014). Optimization of heterologous expression of the phytase (PPHY) of Pichia anomala in P. pastoris and its applicability in fractionating allergenic glycinin from soy protein. Journal of Industrial Microbiology and Biotechnology, 41, 977–987.

    Article  CAS  Google Scholar 

  18. Joshi, S., & Satyanarayana, T. (2015). Characteristics and applicability of phytase of the yeast Pichia anomala in synthesizing haloperoxidase. Applied Biochemistry and Biotechnology,. doi:10.1007/s12010-015-1650-y.

    Google Scholar 

  19. Kanekiyo, M., et al. (2005). Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant mycobacterium bovis bacille calmette- gue´rin expressing human immunodeficiency virus type 1gag. Journal of Virology, 79, 8716–8723.

    Article  CAS  Google Scholar 

  20. Kanovsky, J. R. (1984). Singlet oxygen production by chloroperoxidase-hydrogen peroxide-halide systems. Journal of Biological Chemistry, 259, 5596–5600.

    Google Scholar 

  21. Kaur, P., Singh, B., Böer, E., Straube, N., Piontek, M., Satyanarayana, T., & Kunze, G. (2010). Pphy—A cell-bound phytase from the yeast Pichia anomala: Molecular cloning of the gene PPHY and characterization of the recombinant enzyme. Journal of Biotechnology, 149, 8–15.

    Article  CAS  Google Scholar 

  22. Kerovuo, J., Lauraeus, M., Nurminen, P., Kalkkinen, N., & Apajalahti, J. (1998). Isolation, characterization, molecular gene cloning and sequencing of a novel phytase from Bacillus subtilis. Applied and Environmental Microbiology, 64, 2079–2085.

    CAS  Google Scholar 

  23. Komissarov, A. A., Romanova, D. V., & Debabov, V. G. (1995). Complete inactivation of Escherichia coli uridine phosphorylase by modification of asp with Woodwards reagent k. Journal of Biological Chemistry, 270, 10050–10055.

    Article  CAS  Google Scholar 

  24. Konietzny, U., & Greiner, R. (2002). Molecular and catalytic properties of phytate degrading enzymes (phytases). International Journal of Food Science & Technology, 37, 791–812.

    Article  CAS  Google Scholar 

  25. Kumar, V., & Satyanarayana, T. (2013). Biochemical and thermodynamic characteristics of thermo-alkali-stable xylanase from a novel polyextremophilic Bacillus halodurans TSEV1. Extremophiles, 17, 797–808.

    Article  CAS  Google Scholar 

  26. Lammertyn, E., Van, M. L., Bijnens, A. P., Joris, B., & Anné, J. (1996). Codon adjustment to maximise heterologous gene expression in Streptomyces lividans can lead to decreased mRNA stability and protein yield. Molecular and General Genetics, 250, 223–229.

    CAS  Google Scholar 

  27. Lee, C., Lee, S., Shin, S. G., & Hwang, S. (2008). Real-time PCR determination of rRNA gene copy number: Absolute and relative quantification assays with Escherichia coli. Applied Microbiology and Biotechnology, 78, 371–376.

    Article  CAS  Google Scholar 

  28. Lee, S., Kim, T., Stahl, C. H., & Lei, X. G. (2005). Expression of Escherichia coli AppA2 phytase in four yeast systems. Biotechnology Letter, 27, 327–334.

    Article  CAS  Google Scholar 

  29. Liu, S. Y., et al. (2015). Effects of 500 and 1000 FTU/kg phytase supplementation of maize based diets with two tiers of nutrient specifications on performance of broiler chickens. Animal Feed Science and Technology,. doi:10.1016/j.anifeedsci.2015.06.002.

    Google Scholar 

  30. Mansur, M., et al. (2005). Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnology Letter, 27, 339–345.

    Article  CAS  Google Scholar 

  31. Nampoothiri, K. M., Tomes, G. J., Roopesh, K., Szakacs, G., Nagy, V., Soccol, C. R., & Pandey, A. (2004). Thermostable phytase production by Thermoascus aurantiacus in submerged fermentation. Applied Biochemistry and Biotechnology, 118, 205–214.

    Article  CAS  Google Scholar 

  32. Oka, C., et al. (1999). Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris. Bioscience, Biotechnology and Biochemistry, 63, 1977–1983.

    Article  CAS  Google Scholar 

  33. Outchkourov, N. S., Stiekema, W. J., & Jongsma, M. A. (2002). Optimization of the expression of equistatin in Pichia pastoris. Protein Expression and Purification, 24, 18–24.

    Article  CAS  Google Scholar 

  34. Paoli, P., Fiaschi, T., Cirri, P., Camici, G., Manao, G., Cappugi, G., et al. (1997). Mechanism of acylphosphatase inactivation by Woodward’s reagent k. Journal of Biochemistry, 328, 855–861.

    Article  CAS  Google Scholar 

  35. Pasamontes, L., Haiker, M., Wyss, M., Tessier, M., & Van Loon, A. (1997). Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus. Applied and Environmental Microbiology, 63, 1696–1700.

    CAS  Google Scholar 

  36. Raboy, V. (2003). Myo-Inositol-1,2,3,4,5,6-hexakisphosphate. Phytochemistry, 64, 1033–1043.

    Article  CAS  Google Scholar 

  37. 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, 373–378.

    Article  CAS  Google Scholar 

  38. Sariyska, M. V., Gargova, S. A., Koleva, L. A., & Angelov, A. I. (2005). Aspergillus niger phytase: Purification and characterization. Biotechnology and Biotechnological Equipment, 19, 98–105.

    Article  CAS  Google Scholar 

  39. Sato, K., Hamada, M., Asai, K., & Mituyama, T. (2009). Centroidfold: A web server for RNA secondary structure prediction. Nucleic Acids Research, 37, 277–280.

    Article  Google Scholar 

  40. De Schutter, K., Lin, Y. C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J., et al. (2009). Genome sequence of the recombinant protein production host Pichia pastoris. Nature Biotechnology, 27, 561–566.

    Article  Google Scholar 

  41. Sebastian, S., Touchburn, S., Chavez, E., & Lague, P. (1996). Efficacy of supplemental microbial phytase at different dietary calcium levels on growth performance and mineral utilization of broiler chickens. Poultry Science, 75, 1516–1523.

    Article  CAS  Google Scholar 

  42. Selle, P. H., & Ravindran, V. (2007). Microbial phytase in poultry nutrition. Animal Feed Science and Technology, 135, 1–41.

    Article  CAS  Google Scholar 

  43. Sharp, P. M., & Li, W. H. (1987). The codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Research, 15, 1281–1295.

    Article  CAS  Google Scholar 

  44. Sinclair, G., & Choy, F. Y. M. (2002). Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris. Protein Expression and Purification, 26, 96–105.

    Article  CAS  Google Scholar 

  45. Singh, B., & Satyanarayana, T. (2006). A marked enhancement in phytase production by a thermophilic mould Sporotrichum thermophile using statistical designs in a cost-effective cane molasses medium. Journal of Applied Microbiology, 101, 344–352.

    Article  CAS  Google Scholar 

  46. Singh, B., & Satyanarayana, T. (2006). Phytase production by a thermophilic mold Sporotrichum thermophile in solid state fermentation and its application in dephytinization of sesame oil cake. Applied Biochemistry and Biotechnology, 133, 239–250.

    Article  CAS  Google Scholar 

  47. Singh, B., & Satyanarayana, T. (2008). Improved phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation due to statistical designs. Bioresource Technology, 99, 824–830.

    Article  CAS  Google Scholar 

  48. Singh, B., & Satyanarayana, T. (2008). Phytase production by a thermophilic mould Sporotrichum thermophile in solid state fermentation and its potential applications. Bioresource Technology, 99, 2824–2830.

    Article  CAS  Google Scholar 

  49. Singh, B., & Satyanarayana, T. (2008). Phytase production by a thermophilic mould Sporotrichum thermophile in cost-effective cane molasses medium and its application in bread. Journal of Applied Microbiology, 105, 1858–1865.

    Article  CAS  Google Scholar 

  50. Singh, B., & Satyanarayana, T. (2009). Characterization of a HAP-phytase from a thermophilic mould Sporotrichum thermophile. Bioresource Technology, 100, 2046–2051.

    Article  CAS  Google Scholar 

  51. Sreekrishna, K., et al. (1997). Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene, 190, 55–62.

    Article  CAS  Google Scholar 

  52. Tuller, T., Waldman, Y. Y., Kupiec, M., & Ruppin, E. (2010). Translation efficiency is determined by both codon bias and folding energy. PNAS USA, 107, 3645–3650.

    Article  CAS  Google Scholar 

  53. Ullah, A. H. J., & Cummins, B. J. (1987). Purification, N-terminal amino acid sequence and characterisation of pH 2.5 optimum acid phosphatase (E.C.3.1.3.2) from Aspergillus ficuum. Preparative Biochemistry, 17, 397–422.

    Article  CAS  Google Scholar 

  54. Ushasree, M. V., Vidya, J., & Pandey, A. (2014). Extracellular expression of a thermostable phytase (phyA) in Kluyveromyces lactis. Process Biochemistry, 49, 1440–1447.

    Article  CAS  Google Scholar 

  55. Vats, P., & Banerjee, U. C. (2005). Biochemical characterisation of extracellular phytase (myo inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem. Journal of Industrial Microbiology and Biotechnology, 32, 141–147.

    Article  CAS  Google Scholar 

  56. Villatte, F., Hussein, A. S., Bachmann, T. T., & Schmid, R. D. (2001). Expression level of heterologous proteins in Pichia pastoris is influenced by flask design. Applied Microbiology and Biotechnology, 55, 463–465.

    Article  CAS  Google Scholar 

  57. Vohra, A., & Satyanarayana, T. (2003). Phytases: Microbial sources, production, purification, and potential biotechnological applications. Critical Reviews in Biotechnology, 23, 29–60.

    Article  CAS  Google Scholar 

  58. Wang, Y., Gao, X., Su, Q., Wu, W., & An, L. (2007). Cloning, expression, and enzyme characterization of an acid heat-stable phytase from Aspergillus fumigatus WY-2. Current Microbiology, 55, 65–70.

    Article  CAS  Google Scholar 

  59. Woo, J. H., et al. (2002). Gene optimization is necessary to express a bivalent anti-humananti-T cell immunotoxin in Pichia pastoris. Protein Expression and Purification, 25, 270–282.

    Article  CAS  Google Scholar 

  60. Wyss, M., Pasamontes, L., Remy, R., Kohler, J., Kusznir, E., Gradient, M., et al. (1998). Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger pH 2.5 acid phosphatase. Applied and Environmental Microbiology, 64, 4446–4451.

    CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful to the Department of Biotechnology and University Grant Commission, Govt. of India, New Delhi, for financial assistance and fellowship during the course of this investigation.

Author information

Authors and Affiliations

  1. Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Bibhuti Ranjan & T. Satyanarayana

Authors
  1. Bibhuti Ranjan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T. Satyanarayana
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T. Satyanarayana.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 108 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ranjan, B., Satyanarayana, T. Recombinant HAP Phytase of the Thermophilic Mold Sporotrichum thermophile: Expression of the Codon-Optimized Phytase Gene in Pichia pastoris and Applications. Mol Biotechnol 58, 137–147 (2016). https://doi.org/10.1007/s12033-015-9909-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-015-9909-7

Keywords

Search

Navigation