A Thermostable Xylanase from Clostridium thermocellum Expressed at High Levels in the Apoplast of Transgenic Tobacco Has No Detrimental Effects and Is Easily Purified

Abstract

We expressed a truncated version of the Clostridium thermocellum xylanase (xynZ) gene in transgenic tobacco plants. High levels of the 37 kD protein were synthesized and correctly targeted to the intercellular space by means of the proteinase inhibitor II signal peptide. The protein was one of the most abundant proteins in total extracts that were not protected against proteolysis. Enzyme extracted from leaves retained its activity and hydrolyzed xylan efficiently to xylo-oligomers and xylose. Enzymatic activity could be enriched about 14 to 31-fold after heat-treatment with essentially complete recovery. The transgenic plants, grown under greenhouse conditions, were not affected by the foreign enzyme, possibly due to the high temperature optimum (70°C) of the xylanase and low levels of xylan in dicotyledons. These plants might be useful for production of the enzyme which has numerous applications hi the paper industry and agriculture.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Timell, T.E. 1967. Recent progress in the chemistry of wood hemicelluloses. Wood Sci. Technol. 1: 45–70.

    CAS  Article  Google Scholar 

  2. 2

    McNeil, M., Darvill, A.G., Fry, S.C. and Albersheim, P. 1984. Structure and function of the primary cell walls of plants. Annu. Rev. Biochem. 53: 625–663.

    CAS  Article  Google Scholar 

  3. 3

    Biely, P. 1985. Microbial xylanolytic systems. Trends in Biotechnology 3: 286–290.

    CAS  Article  Google Scholar 

  4. 4

    Wong, K.K.Y., Tan, L.U.L. and Saddler, J.N. 1988. Multiplicity of β-1,4-xylanase in microorganisms: Functions and applications. Microbiol. Reviews 52: 305–317.

    CAS  Google Scholar 

  5. 5

    Grépinet, O., Chebrou, M.-C. and Béguin, P. 1988. Purification of Clostridium themocellum Xylanase Z expressed in Escherichia coli and identification of the corresponding product in the culture medium of C. thermocettum . J. Bacteriol. 170: 4576 4581.

    Article  Google Scholar 

  6. 6

    Grépinet, O., Chebrou, M.-C. and Bégum, P. 1988. Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum . J. Bacteriol. 170: 4582 4588.

    Article  Google Scholar 

  7. 7

    Höfgen, R. and Willmitzer, L. 1990. Biochemical and genetic analysis of different patatin isoforms expressed in various organs of potato (Solomon tuberosum L.). Plant Sci. 66: 221–230.

    Article  Google Scholar 

  8. 8

    von Schaewen, A., Stitt, M., Schmidt, R., Sonnewald. U. and Willmitzer, L. 1990. Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants. EMBO J. 9: 3033–3044.

    CAS  Article  Google Scholar 

  9. 9

    Thomas, J.R., McNeil, M., Darvill, A.G. and Albersheim, P. 1987. Structure of plant cell walls. XIX. Isolation and characterization of wall polysaccharides from suspension-cultured douglas fir cells. Plant Physiol 83: 659–671.

    CAS  Article  Google Scholar 

  10. 10

    Rosahl, S., Schmidt, R., Schell, J. and Willmitzer, L. 1987. Expression of a tuber-specific storage protein in transgenic tobacco plants: demonstration of an esterase activity. EMBO J. 6: 1155–1159.

    CAS  Article  Google Scholar 

  11. 11

    Bradford, M.M. 1976. Rapid and quantitative method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–252.

    CAS  Article  Google Scholar 

  12. 12

    Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350–356.

    CAS  Article  Google Scholar 

  13. 13

    Laliberté, J.-E., Nicolas, O., Durand, S. and Morosoli, R. 1992. The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants. Plant Mol. Biol. 18: 447–451.

    Article  Google Scholar 

  14. 14

    Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Har bor, New York

    Google Scholar 

  15. 15

    Deblaere, R., Bytebier, B., de Greve, H., Debroeck, F.M., Schell, J., van Montagu, M. and Leemanns, J. 1985. Efficient octopine Ti-plasmid derived vectors for Agrobacterium-mediated gene transfer. Nucleic Acids Res. 13: 4777–4788.

    CAS  Article  Google Scholar 

  16. 16

    Royer, J.C. and Nakas, J.P. 1990. Simple, sensitive zymogram technique for detection of xylanase activity in polyacrylamide gels. App. Environ. Microbiol. 56: 1516–1517.

    CAS  Google Scholar 

  17. 17

    Logemann, J., Schell, J. and Willmitzer, L. 1987. Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163: 21–26.

    Article  Google Scholar 

  18. 18

    Amasino, R.M. 1986. Acceleration of nucleic acid hybridisation rate by poly ethylene glycol. Anal. Biochem. 152: 304–307.

    CAS  Article  Google Scholar 

  19. 19

    Laemmli, U.K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685.

    CAS  Article  Google Scholar 

  20. 20

    Biely, P., Mislovicova, D. and Toman, R. 1988. Remazol Brilliant Blue-Xylan: A soluble chromogenic substrate for xylananases. Methods Enzymol. 160: 536–542.

    CAS  Article  Google Scholar 

  21. 21

    Chaplin, M.F. 1986. Monosaccharides, p. 3. In: Carbohydrate Analysis a Practical Approach. Chaplin, M.F. and Kennedy, J.F. (Eds.). IRL Press, Oxford.

    Google Scholar 

Download references

Author information

Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Herbers, K., Wilke, I. & Sonnewald, U. A Thermostable Xylanase from Clostridium thermocellum Expressed at High Levels in the Apoplast of Transgenic Tobacco Has No Detrimental Effects and Is Easily Purified. Nat Biotechnol 13, 63–66 (1995). https://doi.org/10.1038/nbt0195-63

Download citation

Further reading