Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

Isolation, Crystallization and Properties of a New Cellulase of Clostridium thermocellum Overproduced in Escherichia coli

Bio/Technology volume 4, pages 896–900 (1986)Cite this article

Abstract

Subcloning a Clostridium thermocellum DNA fragment carrying the celD gene resulted in the high–level expression in Escherichia coli of a new cellulase termed endoglucanase D. With carboxymethylcellulose as a substrate, endoglucanase D appears to be among the most active cellulases hitherto described. The enzyme was purified from cytoplasmic granules, and crystals suitable for high–resolution X–ray analysis were obtained.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Millet, J., Pétré, D. Béguin, P., Raynaud, O., and Aubert, J.-P. 1985. Cloning of ten distinct DNA fragments of Clostridium thermocellum coding for cellulases. FEMS Microbiol. Lett. 29:145–149.

    Article  CAS  Google Scholar 

  2. Reese, E.T. and Mandels, M. 1984. Rolling with the times. Production and applications of Trichoderma reesei cellulase, p. 1–20. In: Annual Reports on Fermentation Processes. Vol. 7. G. T. Tsao (ed.). Academic Press, Inc., New York.

    Google Scholar 

  3. Van Tilbeurgh, H., Claeyssens, M., and De Bruyne, C.K. 1982. The use of 4-methylumbelliferyl and other chromophoric glycosides in the study of cellulolytic enzymes. FEBS Lett. 149:152–156.

    Article  CAS  Google Scholar 

  4. Pétre, J., Longin, R., and Millet, J. 1981. Purification and properties of an endo-β-1,4-glucanase from Clostridium thermocellum . Biochimie 63:629–639.

    Article  Google Scholar 

  5. Cornet, P., Millet, J., Béguin, P., and Aubert, J.-P. 1983. Characterization of two cel (cellulose degradation) genes of Clostridium thermocellum coding for endoglucanases. Bio/Technology 1:589–594.

    CAS  Google Scholar 

  6. Béguin, P., Cornet, P., and Aubert, J.-P. 1985. Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum . J. Bacteriol. 162:102–105.

    PubMed  PubMed Central  Google Scholar 

  7. Béguin, P., Rocancourt, M., Chebrou, M.-C., and Aubert, J.-P. 1986. Mapping of mRNA encoding endoglucanase A from Clostridium thermocellum . Molec. Gen. Genet. 202:251–254.

    Article  Google Scholar 

  8. Béguin, P., Cornet, P., and Millet, J. 1983. Indentification of the endoglucanase encoded by the celB gene of Clostridium thermocellum . Biochimie 65:495–500.

    Article  Google Scholar 

  9. Grépinet, O. and Béguin, P. 1986. Sequence of the cellulase gene of Clostridium thermocellum coding for endoglucanase B. Nucleic Acids Res. 14:1791–1799.

    Article  Google Scholar 

  10. Pétré, D., Millet, J., Longin, R., Béguin, P., Girard, H., and Aubert, J.-P. 1986. Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli . Biochemie. 68:687–695.

    Article  Google Scholar 

  11. Béguin, P. 1983. Detection of cellulase activity in polyacrylamide gels using Congo red stained agar replicas. Analyt. Biochem. 131:333–336.

    Article  Google Scholar 

  12. Joliff, G., Béguin, P., Millet, J., Aubert, J.P., Alzari, P., Juy, M., and Poljak, R.J. 1986. Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum . J. Mol. Biol. 89:249–250.

    Article  Google Scholar 

  13. Ng, T.K. and Zeikus, J.G. 1981. Purification and characterization of an endogiucanase (1, 4-β-D glucan glucanohydrolase) from Clostridium thermocellum . Biochem. J. 199:341–350.

    Article  CAS  Google Scholar 

  14. Creuzet, N. and Frixon, C. 1983. Purification and characterization of an endoglucanase from a newly isolated thermophilic anaerobic bacterium. Biochimie 65:149–156.

    Article  CAS  Google Scholar 

  15. Deshpande, M.V., Eriksson, K.E., and Pettersson, L.G. 1984. An assay for selective determination of exo-1,4-β-glucanases in a mixture of cellulolytic enzymes. Anal. Biochem. 138:481–487.

    Article  CAS  Google Scholar 

  16. Fukumori, F., Kudo, T., and Horikoshi, K. 1986. Purification and properties of a cellulase from alkalophilic Bacillus sp. N°1139. J. Gen. Microbiol. in press.

  17. Gribskov, M. and Burgess, R.R. 1983. Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene 26:109–118.

    Article  CAS  Google Scholar 

  18. Devos, R., Plaetinck, G., Cheroutre, H., Simons, G., Degrave, W.W., Tavernier, J., Remaut, E., and Fiers, W. 1983. Molecular cloning of human interleukin 2 cDNA and its expression in E. coli . Nucleic Acids Res. 11:4307–4323.

    Article  CAS  Google Scholar 

  19. Simons, G., Remaut, E., Allet, B., Devos, R., and Fiers, W. 1984. High level expression of human interferon gamma in Escherichia coli under control of the PL promoter of bacteriophage lambda. Gene 28:55–64.

    Article  CAS  Google Scholar 

  20. Masui, Y., Mizuno, T., and Inouye, M. 1984. Novel high-level expression cloning vehicles: 104-fold amplification of Escherichia coli minor protein. Bio/Technology 2:81–85.

    CAS  Google Scholar 

  21. Schoner, R.G., Ellis, L.F. and Schoner, B.E. 1985. Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Bio/Technology 3:151–154.

    Article  CAS  Google Scholar 

  22. Casadaban, M.C. and Cohen, S.N. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli . J. Mol. Biol. 138:179–207.

    Article  CAS  Google Scholar 

  23. Messing, J., Crea, R., and Seeburg, P.H. 1981. A system for shotgun DNA sequencing. Nucleic Acids Res. 9:309–321.

    Article  CAS  Google Scholar 

  24. Wain-Hobson, S., Sonigo, P., Danos, O., Cole, S., and Alison, M. 1985. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9–17.

    Article  CAS  Google Scholar 

  25. Vieira, J. and Messing, J. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268.

    Article  CAS  Google Scholar 

  26. Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular cloning, a laboratory manual. Cold Spring Harbor, New York.

    Google Scholar 

  27. Ryter, A. and Kellenberger, E. 1958. Etude au microscope électronique de plasmas contenant de l'cide désoxyribonucléique. I. Les nucléotides de bactéries en croissance. Z. Naturforsch. 13B:597–605.

    Article  CAS  Google Scholar 

  28. Quiviger, B., Franche, C., Lutfalla, G., Rice, D., Haselkorn, R., and Elmerich, C. 1982. Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense . Biochimie 64:495–502.

    Article  CAS  Google Scholar 

  29. Humphreys, G.O., Willshaw, G.A., and Anderson, E.S. 1975. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. Biophys. Acta 382:457–463.

    Article  Google Scholar 

  30. Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523.

    Article  CAS  Google Scholar 

  31. Cohen, S.N., Chang, A.C.Y., and Hsu, L. 1972. Non chromosomal antibiotic resistance in bacteria; genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69:2110–2114.

    Article  CAS  Google Scholar 

  32. Rigby, P.W.J., Dieckmann, M., Rhodes, C., and Berg, P. 1977. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237–251.

    Article  CAS  Google Scholar 

  33. Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503–517.

    Article  CAS  Google Scholar 

  34. Wood, T.M. 1971. The cellulase of Fusarium solani . Biochem. J. 121:353–362.

    Article  CAS  Google Scholar 

  35. Wlodawer, A. and Hodgson, K.O. 1975. Crystallization and crystal data of monellin. Proc. Natl. Acad. Sci. USA 72:398–399.

    Article  CAS  Google Scholar 

  36. Cornet, P., Tronik, D., Millet, J., and Aubert, J.-P. 1983. Cloning and expression in Escherichia coli of Clostridium thermocellum genes coding for amino acid synthesis and cellulose hydrolysis. FEMS Microbiol. Lett. 16:137–141.

    Article  CAS  Google Scholar 

  37. Thomas, R. 1956. Fungal cellulases. Austral. J. Biol. Sci. 9:159–183.

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Michel Juy and Roberto Poljak: Unité d'Immunologie Structurale, Institut Pasteur, 75724 Paris Cedex 15, France.

  2. Antoinette Ryter: Unité de Microscopie Electronique, Institut Pasteur, 75724 Paris Cedex 15, France.

Authors and Affiliations

  1. Unité de Physiologie Cellulaire, Institut Pasteur, 75724, Paris, Cedex 15, France

    Gwennaël Joliff, Pierre Béguin, Jacqueline Millet & Jean-Paul Aubert

Authors
  1. Gwennaël Joliff
    View author publications

    Search author on:PubMed Google Scholar

  2. Pierre Béguin
    View author publications

    Search author on:PubMed Google Scholar

  3. Michel Juy
    View author publications

    Search author on:PubMed Google Scholar

  4. Jacqueline Millet
    View author publications

    Search author on:PubMed Google Scholar

  5. Antoinette Ryter
    View author publications

    Search author on:PubMed Google Scholar

  6. Roberto Poljak
    View author publications

    Search author on:PubMed Google Scholar

  7. Jean-Paul Aubert
    View author publications

    Search author on:PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Joliff, G., Béguin, P., Juy, M. et al. Isolation, Crystallization and Properties of a New Cellulase of Clostridium thermocellum Overproduced in Escherichia coli. Nat Biotechnol 4, 896–900 (1986). https://doi.org/10.1038/nbt1086-896

Download citation

  • Received:

  • Accepted:

  • Issue date:

  • DOI: https://doi.org/10.1038/nbt1086-896

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing