Skip to main content
SpringerLink
Log in

Influence of Linker Length Variations on the Biomass-Degrading Performance of Heat-Active Enzyme Chimeras

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Plant cell walls are composed of complex polysaccharides such as cellulose and hemicellulose. In order to efficiently hydrolyze cellulose, the synergistic action of several cellulases is required. Some anaerobic cellulolytic bacteria form multienzyme complexes, namely cellulosomes, while other microorganisms produce a portfolio of diverse enzymes that work in synergistic fashion. Molecular biological methods can mimic such effects through the generation of artificial bi- or multifunctional fusion enzymes. Endoglucanase and β-glucosidase from extremely thermophilic anaerobic bacteria Fervidobacterium gondwanense and Fervidobacterium islandicum, respectively, were fused end-to-end in an approach to optimize polysaccharide degradation. Both enzymes are optimally active at 90 °C and pH 6.0–7.0 representing excellent candidates for fusion experiments. The direct linkage of both enzymes led to an increased activity toward the substrate specific for β-glucosidase, but to a decreased activity of endoglucanase. However, these enzyme chimeras were superior over 1:1 mixtures of individual enzymes, because combined activities resulted in a higher final product yield. Therefore, such fusion enzymes exhibit promising features for application in industrial bioethanol production processes.

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

Similar content being viewed by others

References

  1. Coughlan, M. P. (1990). Cellulose degradation by fungi. In W. M. Fogarty & C. T. Kely (Eds.), Microbial enzymes and biotechnology (pp. 1–36). London: Elsevier Applied Science.

    Chapter  Google Scholar 

  2. Geddes, C. C., Nieves, I. U., & Ingram, L. O. (2011). Advances in ethanol production. Current Opinion in Biotechnology, 22, 312–319.

    Article  CAS  Google Scholar 

  3. Laursen, W. (2006). Students take a green initiative. The Chemical Engineer, 774–775, 32–34.

    Google Scholar 

  4. Stephanopoulos, G. (2007). Challenges in engineering microbes for biofuels production. Science, 315, 801–804.

    Article  CAS  Google Scholar 

  5. Tollefson, J. (2008). Energy: Not your father’s biofuels. Nature, 451, 880–883.

    Article  CAS  Google Scholar 

  6. Pauly, M., & Keegstra, K. (2008). Cell-wall carbohydrates and their modification as a resource for biofuels. Plant Journal, 54, 559–568.

    Article  CAS  Google Scholar 

  7. Horn, S. J., Vaaje-Kolstad, G., Westereng, B., & Eijsink, V. G. (2012). Novel enzymes for the degradation of cellulose. Biotechnology for Biofuels, 5, 45.

    Article  CAS  Google Scholar 

  8. Liu, L., Wang, L., Zhang, Z., Guo, X., Li, X., & Chen, H. (2012). Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase. BMC Biotechnology, 12, 28.

    Article  CAS  Google Scholar 

  9. Haki, G. D., & Rakshit, S. K. (2003). Developments in industrially important thermostable enzymes: A review. Bioresource technology, 89, 17–34.

    Article  CAS  Google Scholar 

  10. Talebnia, F., Karakashev, D., & Angelidaki, I. (2010). Production of bioethanol from wheat straw: An overview on pretreatment, hydrolysis and fermentation. Bioresource technology, 101, 4744–4753.

    Article  CAS  Google Scholar 

  11. Bhalla, A., Bansal, N., Kumar, S., Bischoff, K. M., & Sani, R. K. (2013). Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresource technology, 128, 751–759.

    Article  CAS  Google Scholar 

  12. Elleuche, S., Schröder, C., Sahm, K., & Antranikian, G. (2014). Extremozymes–biocatalysts with unique properties from extremophilic microorganisms. Current Opinion in Biotechnology, 29, 116–123.

    Article  CAS  Google Scholar 

  13. Viikari, L., Alapuranen, M., Puranen, T., Vehmaanpera, J., & Siika-Aho, M. (2007). Thermostable enzymes in lignocellulose hydrolysis. Advances in Biochemical Engineering/Biotechnology, 108, 121–145.

    Article  CAS  Google Scholar 

  14. Adlakha, N., Rajagopal, R., Kumar, S., Reddy, V. S., & Yazdani, S. S. (2011). Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect gut bacterium. Applied and Environmental Microbiology, 77, 4859–4866.

    Article  CAS  Google Scholar 

  15. Elleuche, S. (2015). Bringing functions together with fusion enzymes-from nature’s inventions to biotechnological applications. Applied Microbiology Biotechnology, 99, 1545–1556.

    Article  CAS  Google Scholar 

  16. Fujita, Y., Ito, J., Ueda, M., Fukuda, H., & Kondo, A. (2004). Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Applied and Environmental Microbiology, 70, 1207–1212.

    Article  CAS  Google Scholar 

  17. Jeon, E., Hyeon, J., Eun, L. S., Park, B. S., Kim, S. W., Lee, J., & Han, S. O. (2009). Cellulosic alcoholic fermentation using recombinant Saccharomyces cerevisiae engineered for the production of Clostridium cellulovorans endoglucanase and Saccharomycopsis fibuligera beta-glucosidase. FEMS Microbiology Letters, 301, 130–136.

    Article  CAS  Google Scholar 

  18. Neddersen, M., & Elleuche, S. (2015). Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras. AMB Express, 5, 33.

    Article  Google Scholar 

  19. Bommarius, A. S., Sohn, M., Kang, Y., Lee, J. H., & Realff, M. J. (2014). Protein engineering of cellulases. Current Opinion in Biotechnology, 29, 139–145.

    Article  CAS  Google Scholar 

  20. Huang, X., Holden, H. M., & Raushel, F. M. (2001). Channeling of substrates and intermediates in enzyme-catalyzed reactions. Annual Review in Biochemistry, 70, 149–180.

    Article  CAS  Google Scholar 

  21. James, C. L., & Viola, R. E. (2002). Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway. Biochemistry, 41, 3720–3725.

    Article  CAS  Google Scholar 

  22. Marcotte, E. M., Pellegrini, M., Ng, H. L., Rice, D. W., Yeates, T. O., & Eisenberg, D. (1999). Detecting protein function and protein-protein interactions from genome sequences. Science, 285, 751–753.

    Article  CAS  Google Scholar 

  23. Sammond, D. W., Payne, C. M., Brunecky, R., Himmel, M. E., Crowley, M. F., & Beckham, G. T. (2012). Cellulase linkers are optimized based on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation. PLoS ONE, 7, e48615.

    Article  CAS  Google Scholar 

  24. Rizk, M., Antranikian, G., & Elleuche, S. (2012). End-to-end gene fusions and their impact on the production of multifunctional biomass degrading enzymes. Biochemical and Biophysical Research Communications, 428, 1–5.

    Article  CAS  Google Scholar 

  25. Jabbour, D., Klippel, B., & Antranikian, G. (2012). A novel thermostable and glucose-tolerant beta-glucosidase from Fervidobacterium islandicum. Applied Microbiology and Biotechnology, 93, 1947–1956.

    Article  CAS  Google Scholar 

  26. Rizk, M., Elleuche, S., & Antranikian, G. (2015). Generating bifunctional fusion enzymes composed of heat-active endoglucanase (Cel5A) and endoxylanase (XylT). Biotechnology Letters, 37, 139–145.

    Article  CAS  Google Scholar 

  27. Elleuche, S., Fodor, K., von der Heyde, A., Klippel, B., Wilmanns, M., & Antranikian, G. (2014). Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region. Applied Microbiology and Biotechnology, 98, 4041–4051.

    Article  CAS  Google Scholar 

  28. Elleuche, S., Döring, K., & Pöggeler, S. (2008). Minimization of a eukaryotic mini-intein. Biochemical and Biophysical Research Communications, 366, 239–243.

    Article  CAS  Google Scholar 

  29. Elleuche, S., Schäfers, C., Blank, S., Schröder, C., & Antranikian, G. (2015). Exploration of extremophiles for high temperature biotechnological processes. Current Opinion in Microbiology, 25, 113–119.

    Article  CAS  Google Scholar 

  30. Berlin, A., Maximenko, V., Gilkes, N., & Saddler, J. (2007). Optimization of enzyme complexes for lignocellulose hydrolysis. Biotechnology and Bioengineering, 97, 287–296.

    Article  CAS  Google Scholar 

  31. Hong, S. Y., Lee, J. S., Cho, K. M., Math, R. K., Kim, Y. H., Hong, S. J., et al. (2007). Construction of the bifunctional enzyme cellulase-beta-glucosidase from the hyperthermophilic bacterium Thermotoga maritima. Biotechnology Letters, 29, 931–936.

    Article  CAS  Google Scholar 

  32. Pei, J., Pang, Q., Zhao, L., Fan, S., & Shi, H. (2012). Thermoanaerobacterium thermosaccharolyticum beta-glucosidase: A glucose-tolerant enzyme with high specific activity for cellobiose. Biotechnology for Biofuels, 5, 31.

    Article  CAS  Google Scholar 

  33. Fan, Z., Wagschal, K., Lee, C. C., Kong, Q., Shen, K. A., Maiti, I. B., & Yuan, L. (2009). The construction and characterization of two xylan-degrading chimeric enzymes. Biotechnology and Bioengineering, 102, 684–692.

    Article  CAS  Google Scholar 

  34. Adlakha, N., Sawant, S., Anil, A., Lali, A., & Yazdani, S. S. (2012). Specific fusion of beta-1,4-endoglucanase and beta-1,4-glucosidase enhances cellulolytic activity and helps in channeling of intermediates. Applied and Environmental Microbiology, 78, 7447–7454.

    Article  CAS  Google Scholar 

  35. Lee, H. L., Chang, C. K., Teng, K. H., & Liang, P. H. (2011). Construction and characterization of different fusion proteins between cellulases and beta-glucosidase to improve glucose production and thermostability. Bioresource technology, 102, 3973–3976.

    Article  CAS  Google Scholar 

  36. Orita, I., Sakamoto, N., Kato, N., Yurimoto, H., & Sakai, Y. (2007). Bifunctional enzyme fusion of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. Applied Microbiology and Biotechnology, 76, 439–445.

    Article  CAS  Google Scholar 

  37. An, J. M., Kim, Y. K., Lim, W. J., Hong, S. Y., An, C. L., Shin, E. C., et al. (2005). Evaluation of a novel bifunctional xylanase-cellulase constructed by gene fusion. Enzyme and Microbial Technology, 36, 989–995.

    Article  CAS  Google Scholar 

  38. Hong, S. Y., Lee, J. S., Cho, K. M., Math, R. K., Kim, Y. H., Hong, S. J., et al. (2006). Assembling a novel bifunctional cellulase-xylanase from Thermotoga maritima by end-to-end fusion. Biotechnology Letters, 28, 1857–1862.

    Article  CAS  Google Scholar 

  39. Lu, P., & Feng, M. G. (2008). Bifunctional enhancement of a beta-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Applied Microbiology and Biotechnology, 79, 579–587.

    Article  CAS  Google Scholar 

  40. Marquardt, T., von der Heyde, A., & Elleuche, S. (2014). Design and establishment of a vector system that enables production of multifusion proteins and easy purification by a two-step affinity chromatography approach. Journal of Microbiological Methods, 105, 47–50.

    Article  CAS  Google Scholar 

  41. Beckham, G. T., Bomble, Y. J., Matthews, J. F., Taylor, C. B., Resch, M. G., Yarbrough, J. M., et al. (2010). The O-glycosylated linker from the Trichoderma reesei Family 7 cellulase is a flexible, disordered protein. Biophysical Journal, 99, 3773–3781.

    Article  CAS  Google Scholar 

  42. Argos, P. (1990). An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. Journal of Molecular Biology, 211, 943–958.

    Article  CAS  Google Scholar 

  43. Li, G., Huang, Z., Zhang, C., Dong, B. J., Guo, R. H., Yue, H. W., et al. (2015). Construction of a linker library with widely controllable flexibility for fusion protein design. Applied Microbiology and Biotechnology, 100, 215–225.

    Article  Google Scholar 

  44. Bai, Y., & Shen, W. C. (2006). Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization. Pharmaceutical Research, 23, 2116–2121.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

MR received a scholarship from the KAAD (Katholischer Akademischer Ausländer-Dienst). The authors thank Ute Lorenz for excellent technical assistance and Maria Kaczmarzyk and Wei-En James Liang for the help with some experiments.

Author information

Authors and Affiliations

  1. Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, 21073, Hamburg, Germany

    Mazen Rizk, Garabed Antranikian & Skander Elleuche

Authors
  1. Mazen Rizk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Garabed Antranikian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Skander Elleuche
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Skander Elleuche.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 186 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rizk, M., Antranikian, G. & Elleuche, S. Influence of Linker Length Variations on the Biomass-Degrading Performance of Heat-Active Enzyme Chimeras. Mol Biotechnol 58, 268–279 (2016). https://doi.org/10.1007/s12033-016-9925-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-016-9925-2

Keywords

Search

Navigation