Skip to main content
SpringerLink
Log in

Expression, purification and characterization of a phyA m-phyCs fusion phytase

  • Published:
Journal of Zhejiang University SCIENCE B Aims and scope Submit manuscript

Abstract

The phyA m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichia pastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyA m-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 °C and an optimal pH at 5.5∼6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 °C to 95 °C for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoHf), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyA m.

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

Similar content being viewed by others

References

  • André, N., Cherouati, N., Prual, C., Steffan, T., Zeder-Lutz, G., Magnin, T., Pattus, F., Michel, H., Wagner, R., Reinhart, C., 2006. Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen. Protein Science, 15(5):1115–1126. [doi:10.1110/ps.062098206]

    Article  PubMed  Google Scholar 

  • Applegate, T.J., Webel, D.M., Lei, X.G., 2003. Efficacy of a phytase derived from Escherichia coli and expressed in yeast on phosphorus utilization and bone mineralization in turkey poults. Poultry Science, 82(11):1726–1732.

    PubMed  CAS  Google Scholar 

  • Augspurger, N.R., Webel, D.M., Lei, X.G., Baker, D.H., 2003. Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs. J. Anim. Sci., 81(2):474–483.

    PubMed  CAS  Google Scholar 

  • Bitar, K., Reinhold, J.G., 1972. Phytase and alkaline phosphatase activities in intestinal mucosae of rat, chicken, calf, and man. Biochem. Biophys. Acta, 268(2):442–452.

    PubMed  CAS  Google Scholar 

  • Bohn, L., Meyer, A.S., Rasmussen, S.K., 2008. Phytate: impact on environment and human nutrition. A challenge for molecular breeding. J. Zhejiang Univ. Sci. B, 9(3):165–191. [doi:10.1631/jzus.B0710640]

    Article  PubMed  CAS  Google Scholar 

  • Chen, C.C., Wu, P.H., Huang, C.T., Cheng, K.J., 2004. A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase. Enzyme and Microbial Technology, 35(4):315–320. [doi:10.1016/j.enzmictec.2004.05.007]

    Article  Google Scholar 

  • Chen, H., Zhao, H.X., Wang, H.N., 2003. The research of phytase’s characteristies from gene engineering yeast. Microbiology, 30(3):38–41 (in Chinese).

    Google Scholar 

  • Chen, H., Zhao, H.X., Wang, H.N., 2005. Increasing expression level of phytase gene (phyA) in Pichia pastoris by changing rare codons. Chinese Journal of Biochemistry and Molecular Biology, 21(2):171–175 (in Chinese).

    CAS  Google Scholar 

  • Common, F.H., 1989. Biological availability of phosphorus for pigs. Nature, 143:370–380.

    Google Scholar 

  • Cregg, J.M., Higginns, D.R., 1995. Production of foreign proteins in the yeast Pichia pastoris. Can. J. Bot., 73(Suppl.):5891–5987.

    Google Scholar 

  • Han, Y., Lei, X.G., 1999. Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris. Archives of Biochemistry and Biophysics, 364(1):83–90. [doi:10.1006/abbi.1999.1115]

    Article  PubMed  CAS  Google Scholar 

  • Han, Y., Wilson, D.B., Lei, X.G., 1999. Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 65(5):1915–1918.

    PubMed  CAS  Google Scholar 

  • Janne, K., Marko, L., Paivi, N., Nisse, K., Juha, A., 1998. Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Applied and Environmental Microbiology, 64(6):2079–2085.

    Google Scholar 

  • Kim, T., Mullaney, E.J., Porres, J.M., Roneker, K.R., Crowe, S., Rice, S., Ko, T., Ullah, A.H.J., Daly, C.B., Welch, R., Lei, X.G., 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):4397–4403 [doi:10.1128/AEM.02612-05]

    Article  PubMed  CAS  Google Scholar 

  • Kim, T.W., Lei, X.G., 2005. An improved method for a rapid determination of phytase activity in animal feed. J. Anim. Sci., 83:1062–1067.

    PubMed  CAS  Google Scholar 

  • Kim, Y.O., Lee, J.K., Kim, H.K., Yu, J.H., Oh, T.K., 1998. Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its expression in E. coli. FEMS Microbiology Letters, 162(1):185–191. [doi:10.1111/j.1574-6968.1998.tb12997.x]

    Article  PubMed  CAS  Google Scholar 

  • Kim, Y.O., Lee, J.K., Oh, B.C., Oh, T.K., 1999. High level expression of a recombinant thermostable phytase in Bacillus subtilis. Biosci. Biotechnol. Biochem., 63(12):2205–2207. [doi:10.1271/bbb.63.2205]

    Article  CAS  Google Scholar 

  • Liu, B.L., Rafiq, A., Tzeng, Y.M., Rob, A., 1998. The induction and characterization of phytase and beyond. Enzyme and Microbial Technology, 22(5):415–424. [doi:10.1016/S0141-0229(97)00210-X]

    Article  CAS  Google Scholar 

  • Markus, W., Luis, P., Arno, F., Roland, R., Michek, T., Alexandra, K., Anke, M., Martin, L., Line, S., Ursro, T., et al., 1999. Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance. Applied and Environmental Microbiology, 65(2):359–366.

    Google Scholar 

  • Olukosi, O.A., Sands, J.S., Adeola, O., 2007. Supplementation of carbohydrases or phytase individually or in combination to diets for weanling and growing-finishing pigs. J. Anim. Sci., 855(7):1702–1711. [doi:10.2527/jas.2006-709]

    Article  Google Scholar 

  • Reddy, N.R., Sathe, S.K., Salunkhe, D.K., 1982. Phytates in legumes and cereals. Adv. Food Res., 28:1–92.

    PubMed  CAS  Google Scholar 

  • Rodriguez, E., Han, Y., Lei, X.G., 1999. Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon. Biochem. Biophys. Res. Commun., 257(1):117–123. [doi:10.1006/bbrc.1999.0361]

    Article  PubMed  CAS  Google Scholar 

  • 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):373–378. [doi:10.1006/bbrc.2000.2121]

    Article  PubMed  CAS  Google Scholar 

  • Romanos, M., 1995. Advances in the use of Pichia pastor is for high-level expression. Curr. Opin. Biotechnol., 6(5):527–533. [doi:10.1016/0958-1669(95)80087-5]

    Article  CAS  Google Scholar 

  • Rutherfurd, S.M., Chung, T.K., Moughan, P.J., 2002. The effect of microbial phytase on ileal phosphorus and amino acid digestibility in the broiler chicken. Br. Poultry Sci., 43(4):598–606. [doi:10.1080/0007166022000004516]

    Article  CAS  Google Scholar 

  • Tye, A.J., Siu, F.K.Y., Leung, T.Y.C., Lim, B.L., 2002. Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis. Appl. Microbiol. Biotechnol., 59(2):190–197. [doi:10.1007/s00253-002-1033-5]

    Article  PubMed  CAS  Google Scholar 

  • Veum, T.L., Bollinger, D.W., Buff, C.E., Bedford, M.R., 2006. A genetically engineered Escherichia coli phytase improves nutrient utilization, growth performance, and bone strength of young swine fed diets deficient in available phosphorus. J. Anim. Sci., 84:1147–1158.

    PubMed  CAS  Google Scholar 

  • Vuolanto, A., von Weymarn, N., Kerovuo, J., Ojamo, H., Leisola, M., 2001. Phytase production by high cell density culture of recombinant Bacillus subtilis. Biotechnology Letters, 23(10):761–766. [doi:10.1023/A:101036932558]

    Article  CAS  Google Scholar 

  • Wang, H.N., Wu, Q., Zhao, H.X., Zou, L.K., Liu, S.G., 2005. Secretory expression of Bacillus subtilis phytase phyC in Pichia pastoris. Journal of Zheijiang University (Agric. & Life Sci.), 31(5):621–627 (in Chinese).

    CAS  Google Scholar 

  • Wodzinski, R.J., Ullah, A.H., 1996. Phytase. Adv. Appl. Microbiol., 42:263–302.

    Article  PubMed  CAS  Google Scholar 

  • Wu, Q., Wang, H.N., Zhou, L.K., Zhao, H.X., Liu, S.G., 2004a. Expression of phytase phyC from B. subtilis in E. coli and its effect in enzymetic thermostability. High Technology Letters, 14(5):23–27 (in Chinese).

    CAS  Google Scholar 

  • Wu, Q., Wang, H.N., Zou, L.K., Zhao, H.X., Liu, S.G., 2004b. Intrcellular expression of Bacillus subtilis phytase phyC in Pichia patoris. Journal of Jishou University (Natural Science Edition), 25(1):36–41 (in Chinese).

    Google Scholar 

  • Xie, Z.W., Wang, H.N., 2007. Preliminary study on flask fermentation condition of producing phytase by engineering bacterium. China Feed, 3:13–16 (in Chinese).

    Google Scholar 

  • Xiong, A.S., Yao, Q.H., Peng, R.H., Han, P.L., Cheng, Z.M., Li, Y., 2005. High level expression of a recombinant acid phytase gene in Pichia pastoris. Journal of applied Microbiology, 98(2):418–428. [doi:10.1111/j.1365-2672.2004.02476.x]

    Article  PubMed  CAS  Google Scholar 

  • Yoshimasu, M.A., Tanaka, T., Ahn, J.K., Yada, R.Y., 2004. Effect of N-linked glycosylation on the aspartic proteinase poreine pepsin expressed from Pichia pastoris. Glycobiology, 14(5):417–429. [doi:10.1093/glycob/cwh024]

    Article  PubMed  CAS  Google Scholar 

  • 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):3069–3076. [doi:10.1128/AEM.02970-06]

    Article  PubMed  CAS  Google Scholar 

  • Zinin, N.V., Serkina, A.V., Gelfand, M.S., Shevelev, A.B., Sineoky, S.P., 2004. Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus. FEMS Microbiology Letters, 236(2):283–290. [doi:10.1111/j.1574-6968.2004.tb09659.x]

    Article  PubMed  CAS  Google Scholar 

  • Zou, L.K., Wang, H.N., Pan, X., Xie, T., Wu, Q., Xie, Z.W., Zhou, W.R., 2006. Design and expression of s synthetic phyC gene encoding the neutral phytase in Pichia pastoris. Acta Biochimica et Biophysica Sinica, 38(11):803–811. [doi:10.1111/j.1745-7270.2006.00231.x]

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. College of Life Science, Bioengineering Research Center for Animal Disease Prevention and Control, Sichuan University, Chengdu, 610065, China

    Li-kou Zou, Hong-ning Wang, Guo-bao Tian, Zi-wen Xie & Zhi-rong Yang

  2. Faculty of Biotechnology of Dujiangyan Campus, Sichuan Agricultural University, Dujiangyan, 611830, China

    Li-kou Zou

  3. College of Earth Science, Chengdu University of Technology, Chengdu, 610059, China

    Xin Pan

  4. College of Life Science, Sichuan Agricultural University, Ya’an, 625014, China

    Qi Wu, Hui Chen & Tao Xie

Authors
  1. Li-kou Zou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hong-ning Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xin Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guo-bao Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zi-wen Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qi Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hui Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tao Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhi-rong Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hong-ning Wang.

Additional information

Project supported by the National Key Technologies R & D Program of China during the 10th Five-Year Plan Period (No. 2002BA514A-12), the Education Department of Sichuan Province (No. 2006B014) and the innovative Fund for Distinguished Young Scholars of Sichuan Agricultural University, China

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zou, Lk., Wang, Hn., Pan, X. et al. Expression, purification and characterization of a phyA m-phyCs fusion phytase. J. Zhejiang Univ. Sci. B 9, 536–545 (2008). https://doi.org/10.1631/jzus.B0720006

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1631/jzus.B0720006

Key words

CLC number

Search

Navigation