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


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.


Citrobacter freundii Codon optimization Pichia pastoris pH-stable Phytase 



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)


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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

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