Skip to main content
SpringerLink
Log in

Efficient Secretory Overexpression of Endoinulinase in Escherichia coli and the Production of Inulooligosaccharides

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

Abstract

Endoinulinase production was achieved by heteroexpression of endoinulinase-encoding gene from Aspergillus ficuum which is an eukaryotic organism in Escherichia coli BL21 (DE3). Further analysis demonstrated that the native signal peptide existed in inu2 gene lowered the enzyme expression level. To realize extracellular accumulation of target protein and improve its expression level, native signal peptide was substituted with pelB, ompC, and pelB fusing with the native signal peptides; then, the effects on endoinulinase production were investigated. As a result, E. coli A606-3, with replacement of pelB as its signal peptide, showed the highest endoinulinase enzyme activity (75.22 U/mg). Also, it suggested that eukaryotic signal peptides have an inhibition on enzyme expression in prokaryotic organism. Moreover, the condition for inulooligosaccharide (IOS) production from inulin was optimized, and an IOS yield of 94.41 % was achieved under the condition of 15 % (w/v) inulin, purified endoinulinase dosage of 5 U/g inulin, 55 °C, and pH 4.6 for 24 h. The major products of hydrolysis of inulin were identified as DP3 to DP7.

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

Similar content being viewed by others

References

  1. Chi, Z., Chi, Z., Zhang, T., Liu, G., & Yue, L. (2009). Inulinase-expressing microorganisms and applications of inulinases. Applied Microbiology and Biotechnology, 82, 211–220.

    Article  CAS  Google Scholar 

  2. Yun, J., Park, J., Song, C., Lee, C., Kim, J., & Song, S. (2000). Continuous production of inulo-oligosaccharides from chicory juice by immobilized endoinulinase. Bioprocess Engineering, 22, 189–194.

    Article  CAS  Google Scholar 

  3. Uhm, T., Chae, K.-S., Lee, D., Kim, H., Cassart, J., & Vandenhaute, J. (1998). Cloning and nucleotide sequence of the endoinulinase-encoding gene, inu2, from Aspergillus ficuum. Biotechnology Letters, 20, 809–812.

    Article  CAS  Google Scholar 

  4. Vandamme, A.-M., Michaux, C., Mayard, A., & Housen, I. (2013). Asparagine 42 of the conserved endo-inulinase INU2 motif WMNDPN from Aspergillus ficuum plays a role in activity specificity. FEBS Open Bio, 3, 467–472.

    Article  CAS  Google Scholar 

  5. Kim, K.-Y., Nascimento, A. S., Golubev, A. M., Polikarpov, I., Kim, C.-S., Kang, S.-I., & Kim, S.-I. (2008). Catalytic mechanism of inulinase from Arthrobacter sp. S37. Biochemical and Biophysical Research Communications, 371, 600–605.

    Article  CAS  Google Scholar 

  6. Nakamura, T., Kurokawa, T., Nakatsu, S., & Ueda, S. (1978). Crystallization and general properties of an extracellular inulase from aspergillus-sp-studies on microbial inulase. 4. Journal of Agricultural Chemical Society of Japan, 52, 159–166.

    Article  CAS  Google Scholar 

  7. Nakamura, T., Shitara, A., Matsuda, S., Matsuo, T., Suiko, M., & Ohta, K. (1997). Production, purification and properties of an endoinulinase of Penicillium sp. TN-88 that liberates inulotriose. Journal of Fermentation and Bioengineering, 84, 313–318.

    Article  CAS  Google Scholar 

  8. Onodera, S., & Shiomi, N. (1988). Purification and substrate specificity of endo-Typo inulinase from Penicillium purpurogenum. Agricultural and Biological Chemistry, 52, 2569–2576.

    CAS  Google Scholar 

  9. Xiao, R., Tanida, M., & Takao, S. (1989). Purification and some properties of endoinulinase from Chrysosporium pannorum. Journal of Fermentation and Bioengineering, 67, 244–248.

    Article  CAS  Google Scholar 

  10. Kang, S.-I., & Kim, S.-I. (1999). Molecular cloning and sequence analysis of an endo-inulinase gene from Arthrobacter sp. Biotechnology Letters, 21, 569–574.

    Article  CAS  Google Scholar 

  11. Jing, W., Zhengyu, J., Bo, J., & Augustine, A. (2003). Production and separation of exo-and endoinulinase from Aspergillus ficuum. Process Biochemistry, 39, 5–11.

    Article  Google Scholar 

  12. Chen, H.-Q., Chen, X.-M., Li, Y., Wang, J., Jin, Z.-Y., Xu, X.-M., Zhao, J.-W., Chen, T.-X., & Xie, Z.-J. (2009). Purification and characterisation of exo-and endo-inulinase from Aspergillus ficuum JNSP5-06. Food Chemistry, 115, 1206–1212.

    Article  CAS  Google Scholar 

  13. Li, Y., Liu, G.-L., Wang, K., Chi, Z.-M., & Madzak, C. (2012). Overexpression of the endo-inulinase gene from Arthrobacter sp. S37 in Yarrowia lipolytica and characterization of the recombinant endo-inulinase. Journal of Molecular Catalysis B: Enzymatic, 74, 109–115.

    Article  CAS  Google Scholar 

  14. Kim, H.-S., Lee, D. W., Ryu, E. J., Uhm, T.-B., Yang, M.-S., Kim, J. B., & Chae, K.-S. (1999). Expression of the INU2 gene for an endoinulinase of Aspergillus ficuum in Saccharomyces cerevisiae. Biotechnology Letters, 21, 621–623.

    Article  CAS  Google Scholar 

  15. He, M., Wu, D., Wu, J., & Chen, J. (2014). Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production. Journal of Industrial Microbiology and Biotechnology, 41, 105–114.

    Article  CAS  Google Scholar 

  16. Chen, M., Lei, X., Chen, C., Zhang, S., Xie, J., & Wei, D. (2015). Cloning, overexpression, and characterization of a highly active endoinulinase gene from Aspergillus fumigatus Cl1 for production of inulo-oligosaccharides. Applied Biochemistry and Biotechnology, 175, 1153–1167.

    Article  CAS  Google Scholar 

  17. Yun, J., Song, C., Choi, J., Choi, Y., & Song, S. (1999). Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity. Bioprocess Engineering, 21, 101–106.

    CAS  Google Scholar 

  18. Chen, X.-M., Xu, X.-M., Jin, Z.-Y., & Chen, H.-Q. (2012). Expression of an endoinulinase from Aspergillus ficuum JNSP5-06 in Escherichia coli and its characterization. Carbohydrate Polymers, 88, 748–753.

    Article  CAS  Google Scholar 

  19. Mergulhao, F., Summers, D., & Monteiro, G. (2005). Recombinant protein secretion in Escherichia coli. Biotechnology Advances, 23, 177–202.

    Article  CAS  Google Scholar 

  20. Hegde, R. S., & Bernstein, H. D. (2006). The surprising complexity of signal sequences. Trends in Biochemical Sciences, 31, 563–571.

    Article  CAS  Google Scholar 

  21. Page, N., Kluepfel, D., Shareck, F., & Morosoli, R. (1996). Effect of signal peptide alterations and replacement on export of xylanase A in Streptomyces lividans. Applied and Environmental Microbiology, 62, 109–114.

    CAS  Google Scholar 

  22. Sjöström, M., Wold, S., Wieslander, A., & Rilfors, L. (1987). Signal peptide amino acid sequences in Escherichia coli contain information related to final protein localization. A multivariate data analysis. The EMBO Journal, 6, 823.

    Google Scholar 

  23. Choi, J., & Lee, S. (2004). Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied Microbiology and Biotechnology, 64, 625–635.

    Article  CAS  Google Scholar 

  24. Sletta, H., Tøndervik, A., Hakvåg, S., Aune, T. V., Nedal, A., Aune, R., Evensen, G., Valla, S., Ellingsen, T., & Brautaset, T. (2007). The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli. Applied and Environmental Microbiology, 73, 906–912.

    Article  CAS  Google Scholar 

  25. Leonhartsberger, S. (2006). E. coli expression system efficiently secretes recombinant proteins into culture broth. BioProcess International, 4, 64–66.

    Google Scholar 

  26. Humphreys, D. P., Sehdev, M., Chapman, A. P., Ganesh, R., Smith, B. J., King, L. M., Glover, D. J., Reeks, D. G., & Stephens, P. E. (2000). High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5′ end of the coding sequence. Protein Expression and Purification, 20, 252–264.

    Article  CAS  Google Scholar 

  27. Lu, L., Fu, F., Zhao, R., Jin, L., He, C., Xu, L., & Xiao, M. (2014). A recombinant levansucrase from Bacillus licheniformis 8-37-0-1 catalyzes versatile transfructosylation reactions. Process Biochemistry, 49, 1503–1510.

    Article  CAS  Google Scholar 

  28. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.

    Article  CAS  Google Scholar 

  29. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  30. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  31. Box, G. E., & Wilson, K. (1951). On the experimental attainment of optimum conditions. Journal of the Royal Statistical Society: Series B Methodological, 13, 1–45.

    Google Scholar 

  32. Choi, J., Jeong, K., Kim, S., & Lee, S. (2000). Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence. Applied Microbiology and Biotechnology, 53, 640–645.

    Article  CAS  Google Scholar 

  33. Matsumoto, T., Katsura, D., Kondo, A., & Fukuda, H. (2002). Efficient secretory overexpression of Bacillus subtilis pectate lyase in Escherichia coli and single-step purification. Biochemical Engineering Journal, 12, 175–179.

    Article  CAS  Google Scholar 

  34. Stern, B., Olsen, L. C., Tröße, C., Ravneberg, H., & Pryme, I. F. (2007). Improving mammalian cell factories: the selection of signal peptide has a major impact on recombinant protein synthesis and secretion in mammalian cells. Trends in Cell and Molecular Biology, 2, 1–17.

    CAS  Google Scholar 

  35. Kim, S. K., Park, Y. C., Lee, H. H., Jeon, S. T., Min, W. K., & Seo, J. H. (2015). Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli. Biotechnology and Bioengineering, 112, 346–355.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (21390200), the Jiangsu Province Natural Science Foundation for Youths (BK20140940), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, a project supported by Program for New Century Excellent Talents in University, Program for Changjiang Scholars and Innovative Research Team in University (Grant No. 06-A-047).

Author information

Authors and Affiliations

  1. State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People’s Republic of China

    Peipei Wang, Jiangfeng Ma, Yue Zhang, Min Zhang, Mingke Wu, Zhongxue Dai & Min Jiang

  2. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South Road 30#, Nanjing, 211816, People’s Republic of China

    Jiangfeng Ma & Min Jiang

Authors
  1. Peipei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiangfeng Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yue Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Min Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mingke Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhongxue Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Min Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jiangfeng Ma or Min Jiang.

Additional information

Peipei Wang and Jiangfeng Ma These authors equally contributed to this study.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, P., Ma, J., Zhang, Y. et al. Efficient Secretory Overexpression of Endoinulinase in Escherichia coli and the Production of Inulooligosaccharides. Appl Biochem Biotechnol 179, 880–894 (2016). https://doi.org/10.1007/s12010-016-2037-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-2037-4

Keywords

Search

Navigation