Advertisement

Applied Biochemistry and Biotechnology

, Volume 162, Issue 8, pp 2157–2165 | Cite as

High Level Expression of an Acid-Stable Phytase from Citrobacter freundii in Pichia pastoris

  • Wei Zhao
  • Aisheng Xiong
  • Xiaoyan Fu
  • Feng Gao
  • Yongsheng Tian
  • Rihe PengEmail author
Article

Abstract

To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni2+–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg−1, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg−1 min−1, respectively. The enzyme activity was significantly improved by 1 mM of K+, Ca2+, and Mg2+. These characteristics contribute to its potential application in feed industry.

Keywords

Citrobacter freundii Codon optimization Pichia pastoris pH-stable Phytase 

Notes

Acknowledgments

The research was supported by the International Scientific and Technological Cooperation of Shanghai-Canada (Alberta-08540706500); Shanghai Rising-Star Program and Natural Science Foundation (08QH14021), High-tech Research, and Development Program of China (2006AA06Z358; 2008AA10Z401).

Supplementary material

12010_2010_8990_MOESM1_ESM.doc (148 kb)
Fig. 1 Nucleotide sequence of the modified gene and deduced amino acid sequence. The potential N-glycosylation sites are underlined (DOC 148 kb)

References

  1. 1.
    Reddy, N. R., Sathe, S. K., & Salunkhe, D. K. (1982). Phytates in legumes and cereals. Advanced Food Research, 28, 1–92.Google Scholar
  2. 2.
    Bitar, K., & Reinhold, J. G. (1972). Phytase and alkaline phosphate activities in intestinal mucosa of rat, chicken, calf and man. Biochimica et Biophysica Acta, 268, 442–452.Google Scholar
  3. 3.
    Abulkalam, M. S. (2008). Demonizing phytate. Nature Biotechnology, 26, 496–497.Google Scholar
  4. 4.
    Nayani, N. R., & Markakis, P. (1983). Effects of inositol phosphates on mineral utilization. Federation Proceedings, 45, 819–826.Google Scholar
  5. 5.
    Peers, F. G. (1953). The phytase of wheat. Biochemical Journal, 53, 102–110.Google Scholar
  6. 6.
    Yanke, L. J., Bae, H. D., Selinger, L. B., & Cheng, K. J. (1998). Phytase activity of anaerobic ruminal bacteria. Microbiology, 144, 1565–1573.CrossRefGoogle Scholar
  7. 7.
    Mélanie, R., André, A., Patrick, C., Santiago, G., Guy, M., & Hélène, B. (2008). Complete hydrolysis of myo-inositol hexakisphosphate by a novel phytase from Debaryomyces castellii CBS 2923. Applied Microbiology and Biotechnology, 78, 47–53.CrossRefGoogle Scholar
  8. 8.
    Gargova, S., Roshkova, Z., & Vancheva, G. (1997). Screening of fungi for phytase production. Biotechnology Techniques, 11, 221–224.CrossRefGoogle Scholar
  9. 9.
    Vats, P., & Banerjee, U. C. (2002). Studies on the production of phytase by a newly isolated strain of Aspergillus niger van Teigham obtained from rotten wood-logs. Process Biochemistry, 38, 211–217.CrossRefGoogle Scholar
  10. 10.
    Harland, B. F., & Oberleas, D. (1999). Phytic acid complex in feed ingredients. In M. B. Coelho & E. T. Kornegay (Eds.), Phytase in animal nutrition and waste management, 2nd review (pp. 69–76). Mexico: BASF.Google Scholar
  11. 11.
    Pillai, U. P., Manoharan, V., Lisle, A., Li, X., & Bryden, W. (2009). Phytase supplemented poultry diets affect soluble phosphorus and nitrogen in manure and manure-amended soil. Journal of Environmental Quality, 38, 1700–1708.CrossRefGoogle Scholar
  12. 12.
    Stefan, H., Anja, K., Edzard, S., Joerg, B., Markus, L., & Oskar, Z. (2005). Biotechnological production and applications of phytases. Applied Microbiology and Biotechnology, 68, 588–597.CrossRefGoogle Scholar
  13. 13.
    Cereghino, J. L., & Cregg, J. M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 24, 45–66.CrossRefGoogle Scholar
  14. 14.
    Peng, R. H., Xiong, A. S., Li, X., Fan, H. Q., Yao, Q. H., Guo, M. J., et al. (2002). High expression of a heat-stable phytase in Pichia pastoris. Acta Biochimica et Biophysica Sinica, 34, 725–730.Google Scholar
  15. 15.
    Xiong, A. S., Yao, Q. H., Peng, R. H., Zhang, Z., Xu, F., Liu, J. G., et al. (2006). High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris. Applied Microbiology and Biotechnology, 72, 1039–1047.CrossRefGoogle Scholar
  16. 16.
    Sharp, P. M., Tuohy, T. M. F., & Mosurski, K. R. (1986). Codon usage in yeast: Cluster analysis clearly differentiates highly and lowly expression genes. Nucleic Acids Research, 14, 5125–5143.CrossRefGoogle Scholar
  17. 17.
    Xiong, A. S., Yao, Q. H., Peng, R. H., Li, X., Fan, H. Q., Li, Y., et al. (2004). A simple, rapid, high fidelity and cost-effective PCR based two-step DNA synthesis (PTDS) method for long gene sequences. Nucleic Acids Research, 32, e98.CrossRefGoogle Scholar
  18. 18.
    Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). New York: Cold Spring Harbor.Google Scholar
  19. 19.
    Heeft, D. (1995). Phytase activity, vanadate manual method. Gist-brocades method Nr, 61696, 2832.Google Scholar
  20. 20.
    Romanos, M., Scorer, C., Sreekrishna, K., & Clare, J. (1998). The generation of multicopy recombinant strains. In D. R. Higgins & J. M. Cregg (Eds.), Pichia protocols (pp. 55–72). Totowa: Human Press.CrossRefGoogle Scholar
  21. 21.
    Clare, J. J., Rayment, F. B., Ballantine, S. P., Sreekrishna, K., & Romanos, M. A. (1991). High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology, 9, 455–460.CrossRefGoogle Scholar
  22. 22.
    Yi, Z., & Kornegay, E. T. (1996). Sites of phytase activity in the gastrointestinal tract of young pigs. Animal Feed Science and Technology, 61, 361–368.CrossRefGoogle Scholar
  23. 23.
    Chesson, A. (1987). Supplementary enzymes to improve the utilization of pig and poultry diets. In W. Haresign & D. J. A. Cole (Eds.), Recent advances in animal nutrition (pp. 71–89). London: Butterworths.Google Scholar
  24. 24.
    Vohra, A., & Satyanarayana, T. (2003). Phytases: microbial sources, production, purification and potential biotechnological applications. Critical Reviews in Biotechnology, 23, 29–60.CrossRefGoogle Scholar
  25. 25.
    Huang, H. Q., Luo, H. Y., Bai, Y. G., Wang, Y. R., & Yao, B. (2006). Overexpression of Citrobacter braakii phytase with high specific activity in Pichia pastoris. Acta Microbiologica Sinica, 46, 945–950.Google Scholar
  26. 26.
    Huang, H. Q., Shao, N., Wang, Y. R., Luo, H. L., Yang, P. L., Zhou, Z. G., et al. (2009). A novel beta-propeller phytase from Pedobacter nyackensis MJ11 CGMCC 2503 with potential as an aquatic feed additive. Applied Microbiology and Biotechnology, 83, 249–259.CrossRefGoogle Scholar
  27. 27.
    Kim, Y. O., Kim, H. W., Lee, J. H., Kim, K. K., & Lee, S. J. (2006). Molecular cloning of the phytase gene from Citrobacter braakii and its expression in Saccharomyces cerevisiae. Biotechnology Letters, 28, 33–38.CrossRefGoogle Scholar
  28. 28.
    Stahl, C. H., Wilson, D. B., & Lei, X. G. (2003). Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris. Biotechnology Letters, 25, 827–831.CrossRefGoogle Scholar
  29. 29.
    Luo, H. Y., Shi, P. J., Li, J., Wang, Y. R., & Yao, B. (2006). Purification and properties of Citrobacter freundii phytase. Acta Microbiologica Sinica, 46, 139–142.Google Scholar
  30. 30.
    Takashi, W., Hiroko, I., Kazuo, M., Tsutomu, F., & Haruyuki, I. (2009). Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris. Journal of Bioscience and Bioengineering, 108, 225–230.CrossRefGoogle Scholar
  31. 31.
    Wang, H. L., Savain, W., & Hesseltine, C. W. (1980). Phytase of molds used in oriental food fermentation. Journal of Food Science, 45, 1261–1266.Google Scholar
  32. 32.
    Henry, B. M. (1926). The effect of enzyme purity on the kinetics of tryptic hydrolysis. The Journal of General Physiology, 10, 217–225.CrossRefGoogle Scholar
  33. 33.
    Ullah, A. H. J., & Gibson, D. M. (1987). Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: Purification and characterization. Preparative Biochemistry & Biotechnology, 17, 63–91.CrossRefGoogle Scholar
  34. 34.
    Tadashi, N., Tatsuya, T., & Hideharu, A. (1999). Dephosphorylation of phytate by using the Aspegillus niger phytase with a high affinity for phytate. Applied and Environmental Microbiology, 65, 4682–4684.Google Scholar
  35. 35.
    Gu, W. N., Huang, H. Q., Meng, K., Yang, P. L., Fu, D. W., Wang, Y. R., et al. (2009). Gene cloning, expression, and characterization of a novel phytase from Dickeya paradisiaca. Applied Microbiology and Biotechnology, 157, 113–123.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Wei Zhao
    • 1
  • Aisheng Xiong
    • 1
  • Xiaoyan Fu
    • 1
  • Feng Gao
    • 1
  • Yongsheng Tian
    • 1
  • Rihe Peng
    • 1
    Email author
  1. 1.Biotechnology Research InstituteShanghai Academy of Agricultural SciencesShanghaiChina

Personalised recommendations