Skip to main content
SpringerLink
Log in

Signal Peptide-Independent Secretory Expression and Characterization of Pullulanase from a Newly Isolated Klebsiella variicola SHN-1 in Escherichia coli

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

Abstract

A strain with the power to produce extracellular pullulanase was obtained from the sample taken from a flour mill. By sequencing its 16S rDNA, the isolate was identified as Klebsiella variicola SHN-1. When the gene encoding pullulanase, containing the N-terminal signal sequence, was cloned into Escherichia coli BL21 (DE3), extracellular activity was detected up to 10 U/ml, a higher level compared with the results in published literature. Subsequently, the recombinant pullulanase was purified and characterized. The main end product from pullulan hydrolyzed by recombinant pullulanase was determined as maltotriose with HPLC, and hence, the recombinant pullulanase was identified as type I pullulanase, which could be efficiently employed in starch processing to produce maltotriose with higher purity and even to evaluate the purity of pullulan. To investigate the effect of signal peptide on secretion of the recombinant enzyme, the signal sequence was removed from the constructed vector. However, secretion of pullulanase in E. coli was not influenced, which was seldom reported previously. By localizing the distribution of pullulanase on subcellular fractions, the secretion of recombinant pullulanase in E. coli BL21 (DE3) was confirmed, even from the expression system of nonsecretory type without the assistance of signal peptide.

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
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Matzke, J., Herrmann, A., Schneider, E., & Bakker, E. (2000). FEMS Microbiology Letters, 183, 55–61.

    Article  CAS  Google Scholar 

  2. Albertson, G. D., McHale, R. H., Gibbs, M. D., & Bergquist, P. L. (1997). Biochimica et Biophysica Acta–Gene Structure and Expression, 1354, 35–39.

    Article  CAS  Google Scholar 

  3. Kang, J., Park, K.-M., Choi, K.-H., Park, C.-S., Kim, G.-E., Kim, D., et al. (2011). Enzyme and Microbial Technology, 48, 260–266.

    Article  CAS  Google Scholar 

  4. Takizawa, N., & Murooka, Y. (1985). Applied and Environmental Microbiology, 49, 294–298.

    CAS  Google Scholar 

  5. Gomes, I., Gomes, J., & Steiner, W. (2003). Bioresource Technology, 90, 207–214.

    Article  CAS  Google Scholar 

  6. Shim, J. H., Park, J. T., Hong, J. S., Kim, K. W., Kim, M. J., Auh, J. H., et al. (2009). Journal of Bacteriology, 191, 4835–4844.

    Article  CAS  Google Scholar 

  7. Hii, S. L., Ling, T. C., Mohamad, R., & Ariff, A. B. (2009). The Open Biotechnology Journal, 3, 1–8.

    Article  CAS  Google Scholar 

  8. Singh, R. S., Saini, G. K., & Kennedy, J. F. (2011). Carbohydrate Polymers, 83, 672–675.

    Article  CAS  Google Scholar 

  9. Zhang, H., & Jin, Z. (2011). Carbohydrate Polymers, 83, 865–867.

    Article  CAS  Google Scholar 

  10. Bender, H., & Wallenfels, K. (1961). Biochemische Zeitschrift, 334, 79–95.

    CAS  Google Scholar 

  11. Walker, G. J. (1968). Biochemical Journal, 108, 33–40.

    CAS  Google Scholar 

  12. Melasniemi, H. (1988). Biochemical Journal, 250, 813–818.

    CAS  Google Scholar 

  13. Ramesh, B., Reddy, P. R. M., Seenayya, G., & Reddy, G. (2001). Bioresource Technology, 76, 169–171.

    Article  CAS  Google Scholar 

  14. Kim, C., Choi, H., & Lee, D. (1993). Journal of Industrial Microbiology and Biotechnology, 12, 48–57.

    Article  CAS  Google Scholar 

  15. Hii, L., Rosfarizan, M., Ling, T., & Ariff, A. (2012). Food and Bioprocess Technology, 5, 729–737.

    Google Scholar 

  16. Plant, A. R., Clemens, R. M., Daniel, R. M., & Morgan, H. W. (1987). Applied Microbiology and Biotechnology, 26, 427–433.

    Article  CAS  Google Scholar 

  17. Odibo, F. J. C., & Obi, S. K. C. (1988). Journal of Industrial Microbiology and Biotechnology, 3, 343–350.

    Article  CAS  Google Scholar 

  18. Gantelet, H., & Duchiron, F. (1998). Applied Microbiology and Biotechnology, 49, 770–777.

    Article  CAS  Google Scholar 

  19. Ben Messaoud, E., Ben Ammar, Y., Mellouli, L., & Bejar, S. (2002). Enzyme and Microbial Technology, 31, 827–832.

    Article  CAS  Google Scholar 

  20. Kunamneni, A., & Singh, S. (2006). Enzyme and Microbial Technology, 39, 1399–1404.

    Article  CAS  Google Scholar 

  21. Doman-Pytka, M., & Bardowski, J. (2004). Critical Reviews in Microbiology, 30, 107–121.

    Article  CAS  Google Scholar 

  22. Lee, S. Y. (1996). Trends in Biotechnology, 14, 98–105.

    Article  CAS  Google Scholar 

  23. Zouari Ayadi, D., Ben Ali, M., Jemli, S., Ben Mabrouk, S., Mezghani, M., Ben Messaoud, E., et al. (2008). Applied Microbiology and Biotechnology, 78, 473–481.

    Article  CAS  Google Scholar 

  24. Tomiyasu, K., Yato, K., Yasuda, M., Tonozuka, T., Ibuka, A., & Sakai, H. (2001). Bioscience, Biotechnology, and Biochemistry, 65, 2090–2094.

    Article  CAS  Google Scholar 

  25. Bertoldo, C., Armbrecht, M., Becker, F., Schafer, T., Antranikian, G., & Liebl, W. (2004). Applied and Environmental Microbiology, 70, 3407–3416.

    Article  CAS  Google Scholar 

  26. Sivashanmugam, A., Murray, V., Cui, C., Zhang, Y., Wang, J., & Li, Q. (2009). Protein Science, 18, 936–948.

    Article  CAS  Google Scholar 

  27. Graslund, S., Nordlund, P., Weigelt, J., Hallberg, B. M., Bray, J., Gileadi, O., et al. (2008). Nature Methods, 5, 135–146.

    Article  Google Scholar 

  28. Jana, S., & Deb, J. K. (2005). Applied Microbiology and Biotechnology, 67, 289–298.

    Article  CAS  Google Scholar 

  29. Krehenbrink, M., Chami, M., Guilvout, I., Alzari, P. M., Pecorari, F., & Pugsley, A. P. (2008). Journal of Molecular Biology, 383, 1058–1068.

    Article  CAS  Google Scholar 

  30. Guilvout, I., Chami, M., Berrier, C., Ghazi, A., Engel, A., Pugsley, A. P., et al. (2008). Journal of Molecular Biology, 382, 13–23.

    Article  CAS  Google Scholar 

  31. D’Enfert, C., & Pugsley, A. P. (1989). Journal of Bacteriology, 171, 3673–3679.

    Google Scholar 

  32. Chami, M., Guilvout, I., Gregorini, M., Remigy, H. W., Muller, S. A., Valerio, M., et al. (2005). Journal of Biological Chemistry, 280, 37732–37741.

    Article  CAS  Google Scholar 

  33. Buddelmeijer, N., Francetic, O., & Pugsley, A. P. (2006). Journal of Bacteriology, 188, 2928–2935.

    Article  CAS  Google Scholar 

  34. Francetic, O., Buddelmeijer, N., Lewenza, S., Kumamoto, C. A., & Pugsley, A. P. (2007). Journal of Bacteriology, 189, 1783–1793.

    Article  CAS  Google Scholar 

  35. Buddelmeijer, N., Krehenbrink, M., Pecorari, F., & Pugsley, A. P. (2009). Journal of Bacteriology, 191, 161–168.

    Article  CAS  Google Scholar 

  36. D’Enfert, C., Ryter, A., & Pugsley, A. P. (1987). The EMBO Journal, 6, 3531–3538.

    Google Scholar 

  37. Takizawa, N., & Murooka, Y. (1984). Agricultural and Biological Chemistry, 48, 1451–1458.

    Article  CAS  Google Scholar 

  38. Michaelis, S., Chapon, C., D’Enfert, C., Pugsley, A. P., & Schwartz, M. (1985). Journal of Bacteriology, 164, 633–638.

    CAS  Google Scholar 

  39. Ruijssenaars, H. J., & Hartmans, S. (2001). Applied Microbiology and Biotechnology, 55, 143–149.

    Article  CAS  Google Scholar 

  40. Ueda, S., & Ohba, R. (1972). Agricultural and Biological Chemistry, 36, 2381–2391.

    Article  CAS  Google Scholar 

  41. Yamashita, M., Kinoshita, T., Ihara, M., Mikawa, T., & Murooka, Y. (1994). Journal of Biochemistry, 116, 1233–1240.

    CAS  Google Scholar 

  42. Takizawa, N., Shiro, H., Hatta, T., Nagao, K., & Kiyohara, H. (1991). Agricultural and Biological Chemistry, 55, 1467–1473.

    Article  CAS  Google Scholar 

  43. Wu, S. J., Kim, J. M., Zhou, C. A., Jin, Z. Y., & Tong, Q. Y. (2010). Biotechnology Letters, 32, 1143–1145.

    Article  CAS  Google Scholar 

  44. Singh, R. S., Saini, G. K., & Kennedy, J. F. (2010). Carbohydrate Polymers, 80, 401–407.

    Article  CAS  Google Scholar 

  45. Sauvonnet, N., & Pugsley, A. P. (1996). Molecular Microbiology, 22, 1–7.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Financial support from Hi-Tech Research and Development Program of China (863 Program) (no. 2012AA022207). The National Key Basic Research and Development Program of China (973 Program) (no. 2011CB710800 and 2009CB724706) and the Program of Introducing Talents of Discipline to Universities (111 Project) (111-2-06) is greatly appreciated.

Author information

Authors and Affiliations

  1. School of Biotechnology, Jiangnan University, 1800 Lihu Ave, Wuxi, 214122, Jiangsu Province, China

    Wen-Bo Chen, Yao Nie & Yan Xu

Authors
  1. Wen-Bo Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yao Nie
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yan Xu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, WB., Nie, Y. & Xu, Y. Signal Peptide-Independent Secretory Expression and Characterization of Pullulanase from a Newly Isolated Klebsiella variicola SHN-1 in Escherichia coli . Appl Biochem Biotechnol 169, 41–54 (2013). https://doi.org/10.1007/s12010-012-9948-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-012-9948-5

Keywords

Search

Navigation