Applied Biochemistry and Biotechnology

, Volume 101, Issue 1, pp 41–60 | Cite as

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) of Trichoderma reesei

  • Torny Eriksson
  • Johan Karlsson
  • Folke TjerneldEmail author


It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.

Index Entries

Cellulase cellulose lignocellulose hydrolysis Trichoderma reesei synergism adsorption Cel7A Cel7B 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Eklund, R., Galbe, M., and Zacchi, G. (1995), Biores. Technol. 52, 225–229.CrossRefGoogle Scholar
  2. 2.
    Ohmine, K., Ooshima, H., and Harano, Y. (1983), Biotechnol. Bioeng. 25, 2041–2053.CrossRefGoogle Scholar
  3. 3.
    Desai, S. G. and Converse, A. O. (1997), Biotechnol. Bioeng. 56, 650–655.CrossRefGoogle Scholar
  4. 4.
    Väljamäe, P., Sild, V., Pettersson, G., and Johansson, G. (1998), Eur. J. Biochem. 253, 469–475.PubMedCrossRefGoogle Scholar
  5. 5.
    Zhang, S., Wolfgang, D., and Wilson, D. (1999), Biotechnol. Bioeng. 66, 35–41.PubMedCrossRefGoogle Scholar
  6. 6.
    Mooney, C., Mansfield, S., Touhy, M., and Saddler, J. (1998), Biores. Technol. 64, 113–119.CrossRefGoogle Scholar
  7. 7.
    Holtzapple, M., Cognata, M., Shu, Y., and Hendrickson, C. (1990), Biotechnol. Bioeng. 36, 275–287.CrossRefGoogle Scholar
  8. 8.
    Saloheimo, M., Nakari-Setälä, T., Tenkanen, M., and Penttilä, M. (1997), Eur. J. Biochem. 249, 584–591.PubMedCrossRefGoogle Scholar
  9. 9.
    Teeri, T. T. and Koivula, A. (1995), Carbohydr. Eur. 12, 28–33.Google Scholar
  10. 10.
    Divne, C., Ståhlberg, J., Reinikainen, T., Ruohonen, L., Pettersson, G., Knowles, J. K. C., Teeri, T. T., and Jones, T. A. (1994), Science 265, 524–528.PubMedCrossRefADSGoogle Scholar
  11. 11.
    Kleywegt, G., Zou, J.-Y., Divne, C., Davies, G. J., Sinning, I., Ståhlberg, J., Reinikainen, T., Srisodsuk, M., Teeri, T., and Jones, T. A. (1997), J. Mol. Biol. 272, 383–397.PubMedCrossRefGoogle Scholar
  12. 12.
    Davies, G. (1995), Structure 3, 853–859.PubMedCrossRefGoogle Scholar
  13. 13.
    Stenberg, K., Tengborg, C., Galbe, M., Zacchi, G., Palmqvist, E., and Hahn-Hägerdal, B. (1998), Appl. Biochem. Biotechnol. 70–72, 697–708.CrossRefGoogle Scholar
  14. 14.
    Gregg, D. and Saddler, J. N. (1996), Biotechnol. Bioeng. 51, 375–383.CrossRefGoogle Scholar
  15. 15.
    Ramos, L. P., Filho, Z., Deschamps, F. C., and Saddler, J. N. (1999), Enzyme Microb. Technol. 24, 371–380.CrossRefGoogle Scholar
  16. 16.
    Nidetzky, B. and Claeyssens, M. (1994), Biotechnol. Bioeng. 44, 961–966.CrossRefGoogle Scholar
  17. 17.
    Ståhlberg, J. (1991), Ph.D. Thesis, Uppsala University, Uppsala, Sweden.Google Scholar
  18. 18.
    Henrissat, B., Teeri, T., and Warren, A. (1998), FEBS Lett. 425, 352–354.PubMedCrossRefGoogle Scholar
  19. 19.
    Vrsanská, M. and Biely, P. (1992), Carbohydr. Res. 227, 19–27.CrossRefGoogle Scholar
  20. 20.
    Barr, B. K., Hsieh, Y. L., Ganem, B., and Wilson, D. B. (1996), Biochemistry 35, 586–592.PubMedCrossRefGoogle Scholar
  21. 21.
    Divne, C., Ståhlberg, J., Teeri, T. T., and Jones, T. A. (1998), J. Mol. Biol. 275, 309–325.PubMedCrossRefGoogle Scholar
  22. 22.
    Teeri, T. T., Koivula, A., Linder, M., Wohlfahrt, G., Divne, C., and Jones, T. A. (1998), Biochem. Soc. Trans. 26, 173–178.PubMedGoogle Scholar
  23. 23.
    Imai, T., Boisset, C., Samejima, M., Igarashi, K., and Sugiyama, J. (1998), FEBS Lett. 432, 113–116.PubMedCrossRefGoogle Scholar
  24. 24.
    Teeri, T. (1997), Trends Biotechnol. 15, 160–167.CrossRefGoogle Scholar
  25. 25.
    Stenberg, K., Tengborg, C., Galbe, M., and Zacchi, G. (1998), J. Chem. Technol. Biotechnol. 71, 299–308.CrossRefGoogle Scholar
  26. 26.
    Hägglund, E. (1951), Chemistry of Wood, Academic Press, New York.Google Scholar
  27. 27.
    Rahkamo, L., Siika-aho, M., Vehviläinen, M., Dolk, M., Viikari, L., Nousianen, P., and Buchert, J. (1996), Cellulose 3, 153–163.CrossRefGoogle Scholar
  28. 28.
    Ståhlberg, J., Johansson, G., and Pettersson, G. (1993), Biochim. Biophys. Acta 1157, 107–113.PubMedGoogle Scholar
  29. 29.
    Medve, J., Karlsson, J., Lee, D., and Tjerneld, F. (1998), Biotechnol. Bioeng. 59, 621–634.PubMedCrossRefGoogle Scholar
  30. 30.
    Medve, J., Lee, D., and Tjerneld, F. (1998), J. Chromatogr. 808, 153–165.CrossRefGoogle Scholar
  31. 31.
    Tack, B. T., Dean, J., Eilat, D., Lorenz, P. E., and Schechter, A. N. (1980), J. Biol. Chem. 255, 8842–8847.PubMedGoogle Scholar
  32. 32.
    Karlsson, J., Medve, J., and Tjerneld, F. (1999), Appl. Biochem. Biotechnol. 82, 243–258.PubMedCrossRefGoogle Scholar
  33. 33.
    Tjerneld, F., Persson, I., Albertsson, P., and Hahn-Hägerdal, B. (1985), Biotechnol. Bioeng. Symp. 15, 419–429.Google Scholar
  34. 34.
    Ryu, D. D. Y., Kim, C., and Mandels, M. (1984), Biotechnol. Bioeng. 26, 488–496.CrossRefGoogle Scholar
  35. 35.
    Nidetzky, B., Steiner, W., Hayn, M., and Claeyssens, M. (1994), Biochem. J. 298, 705–710.PubMedGoogle Scholar
  36. 36.
    Väljamäe, P., Sild, V., Nutt, A., Pettersson, G., and Johansson, G. (1999), Eur. J. Biochem. 266, 327–334.PubMedCrossRefGoogle Scholar
  37. 37.
    Karlsson, J. (2000), Ph.D.Thesis, Lund University, Lund, Sweden.Google Scholar
  38. 38.
    Kleman-Leyer, K. M., Siika-aho, M., Teeri, T. T., and Kirk, T. K. (1996), Appl. Environ. Microbiol. 62, 2883–2887.PubMedGoogle Scholar

Copyright information

© Humana Press Inc 2002

Authors and Affiliations

  • Torny Eriksson
    • 1
  • Johan Karlsson
    • 1
  • Folke Tjerneld
    • 1
    Email author
  1. 1.Department of BiochemistryLund UniversityLundSweden

Personalised recommendations