Advertisement

Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca

  • José M. Escovar-KousenEmail author
  • David Wilson
  • Diana Irwin
Article

Abstract

Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material. Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases, which has led to a strong interest in improving their activities. Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called E4). Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose, and good synergistic properties. Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation. In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling. A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model. Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD). The interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve the binding properties of the CBD were selected. Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-α, and the QuickChange mutagenesis method. E. coli BL21-DE3 was used for protein production and expression. The purified proteins were assayed for enzymatic activity on filter paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC). Mutation of the conserved residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose.

Index Entries

Thermonospora fusca Cel9A cellulases protein engineering computer modeling 

References

  1. 1.
    Smith, J. E. (1996), in Biotechnology, Cambridge University Press, Cambridge, MA, pp. 22–23.Google Scholar
  2. 2.
    Walker, L. P., et al. (1993) Biotech. Bioeng. 42, 1019–1028.CrossRefGoogle Scholar
  3. 3.
    Teeri, T. T. (1997), Trends Biotech. 15, 160.CrossRefGoogle Scholar
  4. 4.
    Walker, L. P., et al. (1992), Biotech. Bioeng. 40, 1019–1026.CrossRefGoogle Scholar
  5. 5.
    Irwin, D. C., Spezio, M., Walker, L. P., and Wilson, D. B. (1993), Biotech. Bioeng. 42, 1002–1013.CrossRefGoogle Scholar
  6. 6.
    Davies, G. J., et al. (1996), Acta Crystallographica D52, 7–17.Google Scholar
  7. 7.
    Henrissat, B., et al. (1989), Gene (Amst.) 81, 83–95.Google Scholar
  8. 8.
    Juy, M., et al. (1992), Nature 357, 89–91.CrossRefADSGoogle Scholar
  9. 9.
    Davies, G. J., et al. (1993), Nature 365, 362–364.PubMedCrossRefADSGoogle Scholar
  10. 10.
    Takashima, S., Nakamura, A., Masaki, H., and Uozumi, T. (1996), Biosci. Biotech. Biochem. 60(1), 77–82.CrossRefGoogle Scholar
  11. 11.
    Kraulis, P. J., et al. (1989), Biochemistry 28, 7241–7257.PubMedCrossRefGoogle Scholar
  12. 12.
    Dalbøge, H. and Heldt-Hansen, H. P. (1994), Mol. Gen. Genet. 243, 253–260.PubMedCrossRefGoogle Scholar
  13. 13.
    Azevedo, Mde O., et al. (1990), J. Gen. Microbiol. 136, 120–123.Google Scholar
  14. 14.
    Cui, Z., et al. (1992), Biosci. Biotechnol. Biochem. 56, 1230–1235.PubMedGoogle Scholar
  15. 15.
    Huang, J., et al. (1992), J. Bacteriol. 174, 1314–1323.PubMedGoogle Scholar
  16. 16.
    Ong, E., et al. (1989), Trends Biotechnol. 7, 239–243.CrossRefGoogle Scholar
  17. 17.
    Divne, C., et al. (1994), Science 265, 524–528.PubMedCrossRefADSGoogle Scholar
  18. 18.
    Rouvinen, J., et al. (1990), Science 249, 380–386.PubMedCrossRefADSGoogle Scholar
  19. 19.
    Spezio, M., et al. (1993), Biochemistry 32, 9906–9916.PubMedCrossRefGoogle Scholar
  20. 20.
    Sakon, J., Irwin, D., Wilson, D. B., and Karplus, P., A. (1997), Nat. Struct. Biol. 4, 810–818PubMedCrossRefGoogle Scholar
  21. 21.
    Irwin, D. C., et al. (1998), J. Bacteriol. 180(7), 1709–1714.PubMedGoogle Scholar
  22. 22.
    Sacco, M., Millet, J., and Aubert, J. P. (1984), Ann. Microbiol. (Inst. Pasteur) 135A, 485–488.CrossRefGoogle Scholar
  23. 23.
    Curry, C., et al. (1988), Appl. Environ. Microbiol. 54, 476–484.PubMedGoogle Scholar
  24. 24.
    Konstantinidis, A. K., et al. (1993), Biochem. J. 291, 883.PubMedGoogle Scholar
  25. 25.
    Stemmer, W. P. C. (1994), Nature 370, 389–391.PubMedCrossRefADSGoogle Scholar
  26. 26.
    Stemmer, W. P. C. (1996), Nat. Biotechnol. 14, 315–319.PubMedCrossRefGoogle Scholar
  27. 27.
    Stemmer, W. P. C. (1995), Biotechnology 13, 549–553.CrossRefGoogle Scholar
  28. 28.
    Matsumura, I. and Ellington, A. D. (1996), Nat. Biotechnol. 14, 366.PubMedCrossRefGoogle Scholar
  29. 29.
    Zhang, J. H., Dawes, G., and Stemmer, W. P. C. (1997), Proc. Natl. Acad. Sci. USA 94, 4504–4509.PubMedCrossRefADSGoogle Scholar
  30. 30.
    Cleland, J. L. and Craik, C. (1996), in Protein Engineering Principles and Practice, Wyley-Liss, New York, NY, pp. 22.Google Scholar
  31. 31.
    Taylor, J. S., Teo, B., Wilson, D. B., and Brady, J. W. (1988), Prot. Eng. 8(11), 1145–1152.CrossRefGoogle Scholar
  32. 32.
    Brooks, C. L., Karplus, M. and Pettit, B. M. (1988), in Proteins: A Theoretical Perspective of Dynamics, Structure an Thermodynamics, Adv. Chem. Phys., Vol. 71, Wiley-Interscience, New York, NY.Google Scholar
  33. 33.
    MacKerell, A. D., Bashford, D., et al (1998), J. Physi. Chem. 102, 3586–3616.Google Scholar
  34. 34.
    Palma, R., Zuccato, P., et al. (2000), in Glycosyl Hydrolases in Biomass Conversion. Himmel, M. E., ed., American Chemical Society, Washington, DC.Google Scholar
  35. 35.
    Van Gunsteren, W. F., and Berendsen, H. J. C. (1977) Mol. Physiol. 34(5), 1311–1327.CrossRefGoogle Scholar
  36. 36.
    Bayer, A. E., et al. (1998) in Carhohydrases from Trichoderma reesei and Other Microorganisms. Claeyssens, M., Nerinckx, W., and Piens, K., eds., The Royal Society of Chemistry, pp. 39–65.Google Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • José M. Escovar-Kousen
    • 1
    Email author
  • David Wilson
    • 2
  • Diana Irwin
    • 2
  1. 1.Martek BiosciencesWinchester
  2. 2.Department of BiochemistryCornell UniversityIthaca

Personalised recommendations