Applied Biochemistry and Biotechnology

, Volume 127, Issue 2, pp 95–110 | Cite as

Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus

Fusion with the β-1,4-glycanase (cex) from the cellulolytic bacterium cellulomonas fimi yields a robust, catalytically active product
  • Gilian Duncan
  • Chris A. Tarling
  • Wade H. Bingle
  • John F. Nomellini
  • Mat Yamage
  • Irene R. Dorocicz
  • Stephen G. Withers
  • John Smit
Original Research Articles

Abstract

Immobilized biocatalysts, including particulate enzymes, represent an attractive tool for research and industrial applications because they combine the specificity of native enzymes with the advantage that they can be readily separated from end product and reused. We demonstrated the use of the Caulobacter crescentus surface (S)-layer protein (RsaA) secretion apparatus for the generation of particulate enzymes. Specifically, a candidate protein made previously by fusion of the β-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated. Cex/RsaA cleaved the glycosidic linkage in the artificial substrate p-nitrophenyl-β-d-cellobioside with a KM similar to that of native Cex (1.1 mM for Cex/RsaA vs 0.60 mM for Cex), indicating that the particulate Cex enzyme was able to bind substrate with wild-type affinity. By contrast, the kcat value was significantly reduced (0.08 s−1 for Cex/RsaA vs 15.8 s−1 for Cex)cat, likely owing to the fact that the RsaA C-terminus induces spontaneous unstructured aggregation of the recombinant protein. Here, we demonstrated that not only can an RsaA fusion protein be cheaply produced and purified to a high yield (76 mg/L of dry wt for Cex/RsaA), but it can also be efficiently recycled. The Caulobacter S-layer secretion system therefore offers an attractive new model system for the production of particulate biocatalysts.

Index Entries

Caulobacter surface (S)-layer protein RsaA type I secretion cellulomonas β-1,4-glycanase cellulase Cex cellobiohydrolase crosslinked enzyme crystals particulate enzymes immobilized biocatalysts 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Freeman, A., Abramov, S., and Georgiou, G. (1999), Biotechnol. Bioeng., 62, 155–159.PubMedCrossRefGoogle Scholar
  2. 2.
    Strauss, A., and Gotz, F. (1996), Mol. Microbiol. 21, 491–500.PubMedCrossRefGoogle Scholar
  3. 3.
    Lee, T. S., Turner, M. K., and Lye, G. J. (2002), Biotechnol. Prog. 18, 43–50.MATHPubMedCrossRefGoogle Scholar
  4. 4.
    Hoskin, F. C., Walker, J. E., and Stote, R. (1999), Chem. Biol. Interact 119–120, 439–444.PubMedCrossRefGoogle Scholar
  5. 5.
    Haring, D., and Schreier, P. (1999), Curr. Opin. Chem. Biol. 3, 35–38.PubMedCrossRefGoogle Scholar
  6. 6.
    Margolin, A. L. (1996), Trends Biotechnol. 14, 223–230.CrossRefGoogle Scholar
  7. 7.
    Ellerby, L. M., Nishida, C. R., Nishida, F., Yamanaka, S. A., Dunn, B., Valentine, J. S., and Zink, J. I. (1992), Science 255, 1113–1115.PubMedCrossRefADSGoogle Scholar
  8. 8.
    Shchipunov, Y. A., Karpenko, T. Y., Bakunina, I. Y., Burtseva, Y. V., and Zvyagintseva, T. N. (2004), J. Biochem. Biophys. Methods 58, 25–38.PubMedCrossRefGoogle Scholar
  9. 9.
    Trivedi, B. (1999), Nat. Biotechnol. 17, 339–341.PubMedCrossRefGoogle Scholar
  10. 10.
    Spagna, G., Barbagallo, R. N., Casarini, D., and Pifferi, P. G. (2001), Enzyme Microb. Technol. 28, 427–438.PubMedCrossRefGoogle Scholar
  11. 11.
    Ichijo, H., Nagasawa, J., and Yamauchi, A. (1990), J. Biotechnol. 14, 169–178.PubMedCrossRefGoogle Scholar
  12. 12.
    Pessela, B. C., Mateo, C., Fuentes, M., Vian, A., Garcia, J. L., Carrascosa, A. V., Guisan, J. M., and Fernandez-Lafuente, R. (2004), Biotechnol. Prog. 20, 388–392.PubMedCrossRefGoogle Scholar
  13. 13.
    Min, D. J., Andrade, J. D., and Stewart, R. J. (1999), Anal. Biochem. 270, 133–139.PubMedCrossRefGoogle Scholar
  14. 14.
    Alvaro, G., Fernandez-Lafuente, R., Blanco, R. M., and Guisan, J. M. (1990), Appl. Biochem. Biotechnol. 26, 181–195.PubMedGoogle Scholar
  15. 15.
    Cao, R., Gu, Z., Patterson, G. D., and Armitage, B. A. (2004), J. Am. Chem. Soc. 126, 726, 727.PubMedCrossRefGoogle Scholar
  16. 16.
    Poindexter, J. S. (1981), Microbiol. Rev. 45, 123–179.PubMedGoogle Scholar
  17. 17.
    Shapiro, L. (1976), Annu. Rev. Microbiol. 30, 377–407.PubMedCrossRefGoogle Scholar
  18. 18.
    Smit, J. (1986), in Bacterial Outer Membranes as Model Systems (Inouye, M., ed.), John Wiley & Sons, New York, pp. 343–376.Google Scholar
  19. 19.
    Sleytr, U. B. and Messner, P. (1983), Annu. Rev. Microbiol. 37, 311–339.PubMedCrossRefGoogle Scholar
  20. 20.
    Sara, M. and Sleytr, U. B. (2000), J. Bacteriol. 182, 859–868.PubMedCrossRefGoogle Scholar
  21. 21.
    Sara, M. and Sleytr, U. B. (1996), Prog. Biophys. Mol. Biol. 65, 83–111.PubMedCrossRefGoogle Scholar
  22. 22.
    Messner, P. and Sleytr, U. B. (1992), Adv. Microb. Physiol. 33, 213–275.PubMedCrossRefGoogle Scholar
  23. 23.
    Smit, J., Engelhardt, H., Volker, S., Smith, S. H., and Baumeister, W. (1992), J. Bacteriol. 174 6527–6538.PubMedGoogle Scholar
  24. 24.
    Awram, P. and Smit, J. (1998), J. Bacteriol. 180, 3062–3069.PubMedGoogle Scholar
  25. 25.
    Bingle, W. H., Nomellini, J. F., and Smit, J. (1997), J. Bacteriol. 179, 601–611.PubMedGoogle Scholar
  26. 26.
    Bingle, W. H., Nomellini, J. F., and Smit, J. (2000), J. Bacteriol. 182, 3298–3301.PubMedCrossRefGoogle Scholar
  27. 27.
    Nomellini, J., Toporowski, M. C., and Smit, J. (2004), in Expression Technologies: Current Status and Future Trends (Baneyx, F., ed.), Horizon Scientific Press, Norfolk, UK, pp. 477–524.Google Scholar
  28. 28.
    Nierman, W. C., Feldblyum, T. V., Laub, M. T., et al. (2001), Proc. Natl. Acad. Sci. USA 98, 4136–4141.PubMedCrossRefADSGoogle Scholar
  29. 29.
    Umelo-Njaka, E., Nomellini, J. F., Bingle, W. H., Glasier, L. G., Irvin, R. T., and Smit, J. (2001), Vaccine 19, 1406–1415.PubMedCrossRefGoogle Scholar
  30. 30.
    Umelo-Njaka, E., Bingle, W. H., Borchani, F., Le, K. D., Awram, P., Blake, T., Nomellini, J. F., and Smit, J. (2002), J. Bacteriol. 184, 2709–2718.PubMedCrossRefGoogle Scholar
  31. 31.
    Gilkes, N. R., Claeyssens, M., Aebersold, R., Henrissat, B., Meinke, A., Morrison, H. D., Kilburn, D. G., Warren, R. A., and Miller, R. C. Jr. (1991), Eur. J. Biochem. 202, 367–377.PubMedCrossRefGoogle Scholar
  32. 32.
    White, A., Withers, S. G., Gilkes, N. R., and Rose, D. R. (1994), Biochemistry 33, 12,546–12,552.Google Scholar
  33. 33.
    Tull, D., Withers, S. G., Gilkes, N. R., Kilburn, D. G., Warren, R. A., and Aebersold, R. (1991), J. Biol. Chem. 266, 15,621–15,625.Google Scholar
  34. 34.
    Tull, D., and Withers, S. G. (1994), Biochemistry 33, 6363–6370.PubMedCrossRefGoogle Scholar
  35. 35.
    Notenboom, V., Birsan, C., Nitz, M., Rose, D. R., Warren, R. A., and Withers, S. G. (1998), Nat. Struct. Biol. 5, 812–818.PubMedCrossRefGoogle Scholar
  36. 36.
    Bingle, W. H., Le, K. D., and Smit, J. (1996), Can. J. Microbiol. 42, 672–684.PubMedCrossRefGoogle Scholar
  37. 37.
    Langsford, M. L., Gilkes, N. R., Singh, B., Moser, B., Miller, R. C. Jr., Warren, R. A., and Kilburn, D. G. (1987), FEBS Lett. 225, 163–167.PubMedCrossRefGoogle Scholar
  38. 38.
    Kempton, J. B., and Withers, S. G. (1992), Biochemistry 31, 9961–9969.PubMedCrossRefGoogle Scholar
  39. 39.
    Leatherbarrow, R. J. (1990), Version 2.0 Ed., Erithacus Software, Staines, UK.Google Scholar
  40. 40.
    Kirk, O., Borchert, T. V., and Fuglsang, C. C. (2002), Curr. Opin. Biotechnol. 13, 345–351.PubMedCrossRefGoogle Scholar
  41. 41.
    van Beilen, J. B., and Li, Z. (2002), Curr. Opin. Biotechnol. 13, 338–344.PubMedCrossRefGoogle Scholar
  42. 42.
    Liang, J. F., Li, Y. T., and Yang, V. C. (2000), J. Pharm. Sci. 89, 979–990.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Gilian Duncan
    • 1
  • Chris A. Tarling
    • 2
  • Wade H. Bingle
    • 1
  • John F. Nomellini
    • 1
  • Mat Yamage
    • 1
  • Irene R. Dorocicz
    • 1
  • Stephen G. Withers
    • 2
  • John Smit
    • 1
  1. 1.Department of Microbiology and ImmunologyUniversity of British ColumbiaVancouverCanada
  2. 2.Department of ChemistryUniversity of British ColumbiaVancouverCanada

Personalised recommendations