Skip to main content

Advertisement

Springer Nature Link
Log in

Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR

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

Abstract

The mutant acid phytase (phyA m) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NPep-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyA ep gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower k m value than that of PP-NPm-8 (P < 0.05). In addition, the overall catalytic efficiency (k cat/k m) of the mutant one was 84% higher (P < 0.05) than that of PP-NPm-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency.

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

Access this article

Log in via an institution

Subscribe and save

Springer+
from $39.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

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
Fig. 6
Fig. 7

Similar content being viewed by others

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

References

  1. Gibson, D. M., & Ullah, A. H. (1988). Archives of Biochemistry and Biophysics, 260, 503–513.

    Article  CAS  Google Scholar 

  2. Hegeman, C. E., & Grabau, E. A. (2001). Plant Physiology, 126, 1598–1608.

    Article  CAS  Google Scholar 

  3. Greiner, R., Konietzny, U., & Jany, K. D. (1993). Archives of Biochemistry and Biophysics, 303, 107–113.

    Article  CAS  Google Scholar 

  4. Kim, Y. O., Lee, J. K., Kim, H. K., Yu, J. H., & Oh, T. K. (1998). FEMS Microbiology Letters, 162, 185–191.

    Article  CAS  Google Scholar 

  5. Tseng, Y. H., Fang, T. J., & Tseng, S. M. (2000). Folia Microbiologica, 45, 121–127.

    Article  CAS  Google Scholar 

  6. Ullah, A. H., & Gibson, D. M. (1987). Preparative Biochemistry, 17, 63–91.

    Article  CAS  Google Scholar 

  7. Rodriguez, E., Hanm, Y., & Lei, X. G. (1999). Biochemical and Biophysical Research Communications, 257, 117–123.

    Article  CAS  Google Scholar 

  8. Guo, M. J., Zhuang, Y. P., Chu, J., Zhang, S. L., Xiong, A. S., Peng, R. H., et al. (2007). Process Biochemistry, 42, 1660–1665.

    Article  CAS  Google Scholar 

  9. Mayer, A. F., Hellmuth, K., Schlieker, H., Lopez-Ulibarri, R., Oertel, S., Dahlems, U., et al. (1999). Biotechnology and Bioengineering, 63, 373–381.

    Article  CAS  Google Scholar 

  10. Berka, R. M., Rey, M. W., Brown, K. M., Byun, T., & Klotz, A. V. (1998). Applied and Environmental Microbiology, 64, 4423–4427.

    CAS  Google Scholar 

  11. Pasamontas, L., Haiker, M., Wyss, M., & Van Loon, A. P. (1997). Applied and Environmental Microbiology, 63, 1696–1700.

    Google Scholar 

  12. Mitchell, D. B., Vogel, K., Weimann, B. J., Pasamontes, L., & Van Loon, A. P. G. M. (1997). Microbiology, 143, 245–252.

    Article  CAS  Google Scholar 

  13. Lei, X. G., Ku, P. K., Miller, E. R., Ullrey, D. E., & Yokoyama, M. T. (1993). The Journal of Nutrition, 123, 1117–1123.

    CAS  Google Scholar 

  14. Perney, K. M., Cantor, A. H., Straw, M. L., & Herkelman, K. L. (1993). Poultry Science, 72, 2106–2114.

    CAS  Google Scholar 

  15. Augspurger, N. R., Webel, D. M., Lei, X. G., & Baker, D. H. (2003). Journal of Animal Science, 81, 474–483.

    CAS  Google Scholar 

  16. Olukosi, O. A., Sands, J. S., & Adeola, O. (2007). Journal of Animal Science, 85, 1702–1711.

    Article  CAS  Google Scholar 

  17. Rutherfurd, S. M., Chung, T. K., & Moughan, P. J. (2002). British Poultry Science, 43, 598–606.

    Article  CAS  Google Scholar 

  18. Lehmann, M., Kostrewa, D., Wyss, M., Brugger, R., D’Arcy, A., Pasamontes, L., et al. (2000). Protein Engineering, 13, 49–57.

    Article  CAS  Google Scholar 

  19. Tomschy, A., Brugger, R., Lehmann, M., Svendsen, A., Vogel, K., Kostrewa, D., et al. (2002). Applied and Environmental Microbiology, 68, 1907–1913.

    Article  CAS  Google Scholar 

  20. Kim, M. S., & Lei, X. G. (2008). Applied Microbiology and Biotechnology, 79, 69–75.

    Article  CAS  Google Scholar 

  21. Kim, M. S., Weaver, J. D., & Lei, X. G. (2008). Applied Microbiology and Biotechnology, 79, 751–758.

    Article  CAS  Google Scholar 

  22. Tian, Y. S., Peng, R. H., Xu, J., Zhao, W., Gao, F., Fu, X. Y., et al. (2009). World Journal of Microbiology and Biotechnology, 26, 903–907.

    Article  Google Scholar 

  23. Petrounia, I. P., & Arnold, F. H. (2000). Current Opinion in Biotechnology, 11, 325–330.

    Article  CAS  Google Scholar 

  24. Arnold, F. H. (1993). Current Opinion in Biotechnology, 4, 450–455.

    Article  CAS  Google Scholar 

  25. Kim, J. H., Choi, G. S., Kim, S. B., Kim, W. H., Lee. J. Y., Ryu, Y. W., et al. (2004). Journal of Molecular Catalysis B: Enzymatic, 27, 169–175.

    Google Scholar 

  26. Jones, A., Lamsa, M., Frandsen, T. P., Spendler, T., Harris, P., Sloma, A., et al. (2008). Journal of Biotechnology, 134, 325–333.

    Article  CAS  Google Scholar 

  27. McHunu, N. P., Singh, S., & Permaul, K. (2009). Journal of Biotechnology, 141, 26–30.

    Article  CAS  Google Scholar 

  28. Mullaney, E. J., Daly, C. B., & Ullah, A. H. (2000). Advances in Applied Microbiology, 47, 157–199.

    Article  CAS  Google Scholar 

  29. Wodzinski, R. J., & Ullah, A. H. (1996). Advances in Applied Microbiology, 42, 263–302.

    Article  CAS  Google Scholar 

  30. van Hartingsveldt, W., van Zeijl, C. M., Harteveld, G. M., Gouka, R. J., Suykerbuyk, M. E., Luiten, R. G., et al. (1993). Gene, 127, 87–94.

    Article  Google Scholar 

  31. Wang, H. N., Ma, M. G., Wu, Q., Liu, S. G., & Luo, Y. (2001). Mycosystema, 20, 486–493 (in Chinese).

    Google Scholar 

  32. Wang, H. N., Wu, Q., Liu, S. G., Xie, J., & Ma, M. G. (2001). Wei Sheng Wu Xue Bao, 41, 310–314 (in Chinese).

    CAS  Google Scholar 

  33. Chen, H., Zhao, H. X., & Wang, H. N. (2005). Chinese Journal of Biochemistry and Molecular Biology, 21, 171–175 (in Chinese).

    CAS  Google Scholar 

  34. Tomschy, A., Wyss, M., Kostrewa, D., Vogel, K., Tessier, M., Hofer, S., et al. (2000). FEBS Letters, 472, 169–172.

    Article  CAS  Google Scholar 

  35. Wyss, M., Pasamontes, L., Friedlein, A., Rémy, R., Tessier, M., Kronenberger, A., et al. (1999). Applied and Environmental Microbiology, 65, 359–366.

    CAS  Google Scholar 

  36. Wyss, M., Brugger, R., Kronenberger, A., Rémy, R., Fimbel, R., Oesterhelt, G., et al. (1999). Applied and Environmental Microbiology, 65, 367–373.

    CAS  Google Scholar 

  37. Heinonen, J. K., & Lahti, R. J. (1981). Analytical Biochemistry, 113, 313–317.

    Article  CAS  Google Scholar 

  38. Chomczynski, P. (1992). Analytical Biochemistry, 201, 134–139.

    Article  CAS  Google Scholar 

  39. Lichtenstein, A. V., Moiseev, V. L., & Zaboikin, M. M. (1990). Analytical Biochemistry, 191, 187–191.

    Article  CAS  Google Scholar 

  40. In, M. J., Seo, S. W., Kim, D. C., & Oh, N. S. (2009). Process Biochemistry, 44, 122–126.

    Article  CAS  Google Scholar 

  41. Zou, L. K., Wang, H. N., Pan, X., Tian, G. B., Xie, Z. W., Wu, Q., et al. (2008). Journal of Zhejiang University. Science. B, 9, 536–545.

    Article  CAS  Google Scholar 

  42. Kong, R., Niu, L. X., & Yuan, J. M. (2006). Journal of ShanXi University (Natural Science edition), 29, 425–427 (in Chinese).

    CAS  Google Scholar 

  43. Cadwell, R. C., & Joyce, G. F. (1992). PCR Methods and Applications, 2, 28–33.

    CAS  Google Scholar 

  44. Lin-Goerke, J. L., Robbins, D. J., & Burezak, J. D. (1997). Biotechniques, 23, 409–412.

    CAS  Google Scholar 

  45. Cadwell, R. C., & Joyce, G. F. (1994). PCR Methods and Applications, 3, 136–140.

    Google Scholar 

  46. Moore, J. C., Jin, H. M., Kuchner, O., & Arnold, F. H. (1997). Journal of Molecular Biology, 272, 336–347.

    Article  CAS  Google Scholar 

  47. Vats, P., & Banerjee, U. C. (2005). Journal of Industrial Microbiology and Biotechnology, 32, 141–147.

    Article  CAS  Google Scholar 

  48. Ostanin, K., Harms, E. H., Stevis, P. E., Kuciel, R., Zhou, M. M., & Van Etten, R. L. (1992). The Journal of Biological Chemistry, 267, 22830–22836.

    CAS  Google Scholar 

  49. Kim, T., Mullaney, E. J., Porres, J. M., Roneker, K. R., Crowe, S., Rice, S., et al. (2006). Applied and Environmental Microbiology, 72, 4397–4403.

    Article  CAS  Google Scholar 

  50. Mullaney, E. J., Daly, C. B., Kim, T., Porres, J. M., Lei, X. G., Sethumadhavan, K., et al. (2002). Biochemical and Biophysical Research Communications, 297, 1016–1020.

    Article  CAS  Google Scholar 

  51. Rodriguez, E., Wood, Z. A., Karplus, P. A., & Lei, X. G. (2000). Archives of Biochemistry and Biophysics, 382, 105–112.

    Article  CAS  Google Scholar 

  52. Zhang, W. M., Mullaney, E. J., & Lei, X. G. (2007). Applied and Environmental Microbiology, 73, 3069–3076.

    Article  CAS  Google Scholar 

  53. Xiang, T., Liu, Q., Deacon, A. M., Koshy, M., Kriksunov, I. A., Lei, X. G., et al. (2004). Journal of Molecular Biology, 339, 437–445.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge financial support for this work from the National Natural Science Foundation of China (Grant No. 30671530). The high-throughput screening of mutant library method is on the stage of substantive examination of patent application for invention (Application No. 201010190445.2).

Author information

Authors and Affiliations

  1. College of Life and Physical Science, Sichuan Agricultural University, Ya’an, 625014, China

    Yan Liao, Min Zeng, Zhen-fang Wu, Hui Chen, Qi Wu, Zhi Shan & Xue-yi Han

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

    Hong-ning Wang

Authors
  1. Yan Liao
    View author publications

    Search author on:PubMed Google Scholar

  2. Min Zeng
    View author publications

    Search author on:PubMed Google Scholar

  3. Zhen-fang Wu
    View author publications

    Search author on:PubMed Google Scholar

  4. Hui Chen
    View author publications

    Search author on:PubMed Google Scholar

  5. Hong-ning Wang
    View author publications

    Search author on:PubMed Google Scholar

  6. Qi Wu
    View author publications

    Search author on:PubMed Google Scholar

  7. Zhi Shan
    View author publications

    Search author on:PubMed Google Scholar

  8. Xue-yi Han
    View author publications

    Search author on:PubMed Google Scholar

Corresponding authors

Correspondence to Hui Chen or Hong-ning Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liao, Y., Zeng, M., Wu, Zf. et al. Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR. Appl Biochem Biotechnol 166, 549–562 (2012). https://doi.org/10.1007/s12010-011-9447-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-011-9447-0

Keywords