Skip to main content
SpringerLink
Log in

Expression and Extracellular Secretion of Endo-glucanase and Xylanase by Zymomonas mobilis

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

Abstract

Recombinant Zymomonas mobilis (pGEX-4T-3 BI 120-2) was constructed to encode endo-glucanase (CelA) and endo-xylanase (Xyn11) from Z. mobilis ZM4 (ATCC 31821) and an uncultured bacterium. The recombinant was genetically engineered with the N-terminus of a predicted SecB-dependent (type II) secretion signal from phoC of Z. mobilis to translocate the enzymes extracellularly. Both the enzymes were characterized regarding their functional optimum pH and temperature, with the highest multi-enzyme activities at pH 6.0 and a temperature of 30 °C, which approximates the optimum conditions for ethanol production by Z. mobilis. The crude intracellular and extracellular fractions of the recombinant were characterized in terms of substrate specificity using carboxymethyl cellulose (CMC), beechwood xylan, filter paper, Avicel, and pretreated rice straw. The crude extracellular and intracellular enzymes with cellulolytic and xylanolytic activities were more robustly produced and secreted from the recombinant strain compared to the wild-type and ampicillin-sensitive strains, using CMC and beechwood xylan as the substrates. Ethanol production by the recombinant strain was greater than the production by the wild-type strain when pretreated rice straw was used as a sole carbon source.

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. Lynd, L. R., Weimer, P. J., Van Zyl, W. H., & Pretorius, I. S. (2002). Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and Molecular Biology Reviews, 66(3), 506–577.

    Article  CAS  Google Scholar 

  2. Lynd, L. R., Van Zyl, W. H., McBride, J. E., & Laser, M. (2005). Consolidated bioprocessing of cellulosic biomass: an update. Current Opinion in Biotechnology, 16(5), 577–583.

    Article  CAS  Google Scholar 

  3. Carere, C. R., Sparling, R., Cicek, N., & Levin, D. B. (2008). Third generation biofuels via direct cellulose fermentation. International Journal of Molecular Sciences, 9(7), 1342–1360.

    Article  CAS  Google Scholar 

  4. Antoni, D., Zverlov, V. V., & Schwarz, W. H. (2007). Biofuels from microbes. Applied Microbiology and Biotechnology, 77(1), 23–35.

    Article  CAS  Google Scholar 

  5. Todhanakasem, T., Sangsutthiseree, A., Areerat, K., Young, G. M., & Thanonkeo, P. (2014). Biofilm production by Zymomonas mobilis enhances ethanol production and tolerance to toxic inhibitors from rice bran hydrolysate. New Biotechnology, 31, 451–459.

    Article  CAS  Google Scholar 

  6. Olofsson, K., Bertilsson, M., & Lidén, G. (2008). A short review on SSF—an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnology for Biofuels, 1(1), 7.

    Article  Google Scholar 

  7. Sun, Y., & Cheng, J. (2002). Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology, 83(1), 1–11.

    Article  CAS  Google Scholar 

  8. Rogers, P. L., Jeon, Y. J., Lee, K. J., & Lawford, H. G. (2007). Zymomonas mobilis for fuel ethanol and higher value products. In Biofuels (pp. 263–288). Berlin, Heidelberg: Springer.

  9. Swings, J., & De Ley, J. (1977). The biology of Zymomonas. Bacteriological Reviews, 41, 1.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhang, M., Eddy, C., Deanda, K., Finkelstein, M., & Picataggio, S. (1995). Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science, 267(5195), 240–243.

    Article  CAS  Google Scholar 

  11. Zhang, M., Franden, M., Newman, M., McMillan, J., Finkelstein, M., & Picataggio, S. (1995). Promising ethanologens for xylose fermentation. Applied Biochemistry and Biotechnology, 51, 527–536.

    Article  Google Scholar 

  12. He, M. X., Wu, B., Qin, H., Ruan, Z. Y., Tan, F. R., Wang, J. L., Shui, Z. X., Dai, L. C., Zhu, Q. L., & Pan, K. (2014). Zymomonas mobilis: a novel platform for future biorefineries. Biotechnology for Biofuels, 7, 101.

    Article  CAS  Google Scholar 

  13. Polizeli, M., Rizzatti, A., Monti, R., Terenzi, H., Jorge, J. A., & Amorim, D. (2005). Xylanases from fungi: properties and industrial applications. Applied Microbiology and Biotechnology, 67(5), 577–591.

    Article  CAS  Google Scholar 

  14. Dodd, D., & Cann, I. K. (2009). Enzymatic deconstruction of xylan for biofuel production. GCB Bioenergy, 1(1), 2–17.

    Article  CAS  Google Scholar 

  15. Sanchez, O. J., & Cardona, C. A. (2008). Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresource Technology, 99(13), 5270–5295.

    Article  CAS  Google Scholar 

  16. Linger, J. G., Adney, W. S., & Darzins, A. (2010). Heterologous expression and extracellular secretion of cellulolytic enzymes by Zymomonas mobilis. Applied and Environmental Microbiology, 76, 6360–6369.

    Article  CAS  Google Scholar 

  17. Kojima, M., Okamoto, K., & Yanase, H. (2013). Direct ethanol production from cellulosic materials by Zymobacter palmae carrying Cellulomonas endoglucanase and Ruminococcus β-glucosidase genes. Applied Microbiology and Biotechnology, 97, 5137–5147.

    Article  CAS  Google Scholar 

  18. Luo, Z., & Bao, J. (2015). Secretive expression of heterologous β-glucosidase in Zymomonas mobilis. Bioresources and Bioprocessing, 2, 1–6.

    Article  CAS  Google Scholar 

  19. Seo, J.-S., Chong, H., Park, H. S., Yoon, K.-O., Jung, C., Kim, J. J., Hong, J. H., Kim, H., Kim, J.-H., & Kil, J.-I. (2005). The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. Nature Biotechnology, 23(1), 63–68.

    Article  CAS  Google Scholar 

  20. Rajnish, K., Choudhary, G. K., & Gunasekaran, P. (2008). Functional characterization of a putative endoglucanase gene in the genome of Zymomonas mobilis. Biotechnology Letters, 30(8), 1461–1467.

    Article  CAS  Google Scholar 

  21. Amann, R. I., Ludwig, W., & Schleifer, K.-H. (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiological Reviews, 59(1), 143–169.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Todhanakasem, T., & Jittjang, S. (2016). Evaluation of cellulase production by Zymomonas mobilis. BioResources, 12, 1165–1178.

    Article  Google Scholar 

  23. Stokes, H., Dally, E., Yablonsky, M., & Eveleigh, D. (1983). Comparison of plasmids in strains of Zymomonas mobilis. Plasmid, 9(2), 138–146.

    Article  CAS  Google Scholar 

  24. Cao, Q.-H., Shao, H.-H., Qiu, H., Li, T., Zhang, Y.-Z., & Tan, X.-M. (2017). Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4. Bioscience, Biotechnology, and Biochemistry, 81(3), 453–459.

    Article  CAS  Google Scholar 

  25. Cao, Q., Li, T., Shao, H., Tan, X., & Zhang, Y. (2016). Three new shuttle vectors for heterologous expression in Zymomonas mobilis. Electronic Journal of Biotechnology, 19, 33–40.

    Article  CAS  Google Scholar 

  26. Kanokratana, P., Eurwilaichitr, L., Pootanakit, K., & Champreda, V. (2015). Identification of glycosyl hydrolases from a metagenomic library of microflora in sugarcane bagasse collection site and their cooperative action on cellulose degradation. Journal of Bioscience and Bioengineering, 119(4), 384–391.

    Article  CAS  Google Scholar 

  27. Yanase, H., Kotani, T., & Tonomura, K. (1986). Transformation of Zymomonas mobilis with plasmid DNA. Agricultural and Biological Chemistry, 50, 3139–3144.

    CAS  Google Scholar 

  28. Sandkvist, M. (2001). Biology of type II secretion. Molecular Microbiology, 40(2), 271–283.

    Article  CAS  Google Scholar 

  29. König, J., Grasser, R., Pikor, H., & Vogel, K. (2002). Determination of xylanase, ß-glucanase, and cellulase activity. Analytical and Bioanalytical Chemistry, 374(1), 80–87.

    Article  Google Scholar 

  30. King, F. G., & Hossain, M. A. (1982). The effect of temperature, pH, and initial glucose concentration on the kinetics of ethanol production by Zymomonas mobilis in batch fermentation. Biotechnology Letters, 4(8), 531–536.

    Article  CAS  Google Scholar 

  31. Yanase, H., Nozaki, K., & Okamoto, K. (2005). Ethanol production from cellulosic materials by genetically engineered Zymomonas mobilis. Biotechnology Letters, 27(4), 259–263.

    Article  CAS  Google Scholar 

  32. Imman, S., Arnthong, J., Burapatana, V., Laosiripojana, N., & Champreda, V. (2013). Autohydrolysis of tropical agricultural residues by compressed liquid hot water pretreatment. Applied Biochemistry and Biotechnology, 170(8), 1982–1995.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was financially supported by an Assumption University Research Grant (RP 59-002), Thailand.

Funding

The Z. mobilis ZM4 (NRRL B-14023) used in this study was obtained from Professor Pornthap Thanonkeo, Khon Kaen University, Thailand.

Author information

Authors and Affiliations

  1. Department of Agro-Industry, Faculty of Biotechnology, Assumption University, Ramkhamhaeng Road, Bangkapi, Bangkok, 10240, Thailand

    Tatsaporn Todhanakasem & Apinya Sowatad

  2. National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Phahonyothin Road, Klong Luang, Pathumthani, 12120, Thailand

    Pattanop Kanokratana & Verawat Champreda

  3. Institute of Molecular Biosciences, Mahidol University, Phuttamonthon 4 Road, Salaya, Nakhon Pathom, 73170, Thailand

    Phattara-orn Havanapan

Authors
  1. Tatsaporn Todhanakasem
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Apinya Sowatad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pattanop Kanokratana
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Phattara-orn Havanapan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Verawat Champreda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tatsaporn Todhanakasem.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Todhanakasem, T., Sowatad, A., Kanokratana, P. et al. Expression and Extracellular Secretion of Endo-glucanase and Xylanase by Zymomonas mobilis. Appl Biochem Biotechnol 187, 239–252 (2019). https://doi.org/10.1007/s12010-018-2821-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-018-2821-4

Keywords

Search

Navigation