Applied Biochemistry and Biotechnology

, Volume 22, Issue 1, pp 13–29 | Cite as

Ultraviolet difference spectroscopic study on the interactions of cellulase fromTrichoderma reesei with cellodextrins

  • Wen-Juin Shieh
  • G. J. Tsai
  • M. R. Ladisch
  • G. T. Tsao


The formation of cellodextrin-cellobiohydrolase complex was studied by ultraviolet difference spectroscopy. Upon the binding of cellodextrins (G7‐G3), cellobiohydrolase (EC purified fromTrichoderma reesei produced difference spectra having maxima at 289‐293 nm and 283‐286 nm. These spectra are consistent with prior observations reported for lysozyme and amylase. In this case, water soluble cellulose oligomers (i.e., cellodextrins) are shown to interact with tryptophan residue(s) on cellobiohydrolase. The difference spectral maxima observed at acidic or alkaline pH were shifted. This was accompanied by a marked decrease of binding ability of cellobiohydrolase for cellodextrins. The standard free energy change for the association of cellodextrins to the cellobiohydrolase was an order of 4 kcal/gmol. The association constant of enzyme for substrate decreases by 15‐20% as temperature increases from 20 to 48°C. At 25°C, the dissociation constants for the enzyme with respect to cellohexose and cellotriose were estimated to be 1.19 and 1.37 mM, respectively. A decrease in dissociation constants was observed with an increase in the number of glucosyl units from 3 to 6. This suggests that there may be six or more subsites in the active center of cellobiohydrolase.

Index Entries

Cellulose cellulase cellobiohydrolase cellodextrin(s) difference spectroscopy ultrafiltration subsite degree of polymerization 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Tsao, G. T., Ladisch, M. R., Ladisch, C., Hsu, T. A., Dale, B., and Chou, T. (1978),Ann. Rep. Ferment. Proc. 2, 1–21.Google Scholar
  2. 2.
    Flickinger, M. C. and Tsao, G. T. (1978),Ann. Rep. Ferment. Proc. 2, p. 23.Google Scholar
  3. 3.
    Lipinsky, E. S. (1981),Science 212, p. 1465.CrossRefGoogle Scholar
  4. 4.
    Ladisch, M. R., Lin, K. W., Voloch, M., and Tsao, G. T. (1983),Enzyme Microb. Technol, vol. 5, March.Google Scholar
  5. 5.
    Gong, C. S., Ladisch, M. R., and Tsao, G. T. (1979),Adv. Chem. Ser. (181), p. 261.CrossRefGoogle Scholar
  6. 6.
    Wood, T. M. and McCrae, S. I. (1978),Biochem. J. 171, 61–72.Google Scholar
  7. 7.
    Ryu, D. D. Y., Kim C., and Mandels, M. (1984),Biotechnol. Bioeng. 26, 488–496.CrossRefGoogle Scholar
  8. 8.
    Hayashi, K., Imoto, T., and Funatsu, M. (1963),J. Biochem. 54, 5, 381–387.Google Scholar
  9. 9.
    Mandels, M. and Reese, E. T. (1957),J. Bacteriol. 73, 269–278.CrossRefGoogle Scholar
  10. 10.
    Mandels, M., Andreotti, R., and Roche, C. (1976),Biotechnol. Bioeng. Symp. (6), p. 21.Google Scholar
  11. 11.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951),J. Biol. Chem. 193, 265.Google Scholar
  12. 12.
    Laemmli, U. K. (1970),Nature 227, 680.CrossRefGoogle Scholar
  13. 13.
    Patchornik, A., Lawson, W. G., Gross, E., and Witkop, B. (1960),J. Am. Chem. Soc. 82, 5923–5927.CrossRefGoogle Scholar
  14. 14.
    Miller, G. L., Dean, J., and Blum, R. (1960),Arch. Biochem. Biophys. 91, 21.CrossRefGoogle Scholar
  15. 15.
    Miller, G. L. (1963), inMethods in Carbohydrate Chemistry, vol. 3, Academic Press, New York, 134.Google Scholar
  16. 16.
    Beaven, G. H. and Holiday, E. R. (1952),Adv. Protein Chem. 7, 320.Google Scholar
  17. 17.
    Herokovits, T. T. and Sorensen, M. (1968),Biochemistry 7, 2523–2532.CrossRefGoogle Scholar
  18. 18.
    Collins, K. D. and Stark, G. R. (1969),J. Biol. Chem. 244, 1869–1877.Google Scholar
  19. 19.
    Ohnishi, M. (1970),J. Biochem. 68, 933–936.Google Scholar
  20. 20.
    Clarke, A. J. and Yaguchi, M. (1986),Biochimica et Biophysica Acta 870, 401–407.Google Scholar
  21. 21.
    Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C., and Rupley, J. A. (1972), inThe Enzymes, vol. 7, Boyer, P. D., ed., Academic, New York, 665–868.CrossRefGoogle Scholar
  22. 22.
    French, D. (1981), inBasic Life Sciences, Hollaender, A., et al., eds.), Plenum, New York, vol. 18, 151–182.Google Scholar
  23. 23.
    Hurst, P. L., Nielsen, J., Sullivan, P. A., and Shepherd, M. G. (1977),Biochem. J. 165, 33–41.Google Scholar
  24. 24.
    Hurst, P. L., Sullivan, P. A., and Shepherd, M. G. (1977),Biochem. J. 167, 549–556.Google Scholar
  25. 25.
    Legler, G. and Bause, E. (1973),Carbohydr. Res. 28, 45–52.CrossRefGoogle Scholar
  26. 26.
    Pettersson, G. (1968),Arch. Biochem. Biophys. 126, 776–784.CrossRefGoogle Scholar
  27. 27.
    van Tilbeurgh H., Pettersson, G., Bhikabhai, R., Deboeck, H., and Claeyssens, M. (1985),Eur. J. Biochem. 148, 329–334.CrossRefGoogle Scholar
  28. 28.
    Michaelis, L. and Menten, M. L. (1913),Biochem. Z. 49, 333.Google Scholar
  29. 29.
    Hurst, P. L., Sullivan, P. A., and Shepherd, M. G. (1978),Biochem. J. 169, 389–395.Google Scholar
  30. 30.
    Halliwell, G. and Vincent, R. (1981),Biochem. J. 199, 409–417.Google Scholar
  31. 31.
    Li, L. H., Flora, R. M., and King, K. W. (1965),Arch. Biochem. Biophys. 111, 439–447.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1989

Authors and Affiliations

  • Wen-Juin Shieh
    • 1
  • G. J. Tsai
    • 1
  • M. R. Ladisch
    • 1
  • G. T. Tsao
    • 1
  1. 1.Laboratory of Renewable Resources Engineering and Department of Agricultural EngineeringPurdue UniversityWest Lafayette

Personalised recommendations