Applied Biochemistry and Biotechnology

, Volume 62, Issue 1, pp 15–27 | Cite as

Cloning and expression of the gene for xylose isomerase fromThermus flavus AT62 inEscherichia coli

  • Byoung Chul Park
  • Sukhoon Koh
  • Changsoo Chang
  • Se Won Suh
  • Dae-Sil Lee
  • Si Myung Byun
Original Articles


The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene. The XI gene was stably expressed inE. coli under the control oftac promoter. XI produced inE. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.

Index entries

Thermusflavus AT62 xylose isomerase xylulose kinase overlapping gene gene expression tac promoter 


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  1. 1.
    Danno, G. (1970),Agric. Biol. Chem. 34, 1805–1811.Google Scholar
  2. 2.
    Tasaki, Y., Kosugi, Y., and Kanbayashi, A. (1969),Agric. Biol. Chem. 33, 1527–1535.Google Scholar
  3. 3.
    Yamanaka, K. (1963),Agric. Biol. Chem. 27, 271–279.Google Scholar
  4. 4.
    Szmant, H. H. (1986), InIndustrial Utilization of Renewable Resources: An Introduction. Technomic, Lancaster, PA.Google Scholar
  5. 5.
    Sipos, T. (1973), U.S. Patent 3,708,387.Google Scholar
  6. 6.
    Strandberg, G. W. and Smiley, K. L. (1971),Appl. Microbiol. 21, 588–593.Google Scholar
  7. 7.
    Posno, M., Heuvelmans, P. T. H. M, van Giezen, M. J. F., Lokman, B. C, Leer, R. J., and Pouwels, P. H. (1991),Appl. Environ. Microbiol. 57, 2764–2766.Google Scholar
  8. 8.
    Utt, E. A., Eddy, C. K., Keshav, K. F., and Ingram, L. O. (1991),Appl. Environ. Microbiol. 57, 1227–1234.Google Scholar
  9. 9.
    Schellenberg, G. D., Sarthy, A., Larson, A. E., Backer, M. P., Crabb, J. W., Lidstrom, M., Hall, B. D., and Furlong, C. E. (1984),J. Biol. Chem. 259, 6826–6832.Google Scholar
  10. 10.
    Wilhelm, M. and Hollenberg, C. P. (1985),Nucleic Acids Res. 13, 5717–5722.CrossRefGoogle Scholar
  11. 11.
    Saari, G. C, Kumar, A. A., Kawasaki, G. H., Insley, M. Y., and O’Hara, P. J. (1987),J. Bacteriol. 169, 612–618.Google Scholar
  12. 12.
    Amore, R. and Hollenberg, C. P. (1989),Nucleic Acids Res. 17, 7515.CrossRefGoogle Scholar
  13. 13.
    Dekker, K. A., Yamagata, H., Sakaguchi, K., and Udaka, S. (1991),Agric. Biol. Chem. 55, 221–227.Google Scholar
  14. 14.
    Lee, C, Meng, M., Bagdasarian, M., and Zeikus, J. G. (1990),J. Biol. Chem. 265, 19, 082–19,090.Google Scholar
  15. 15.
    Dekker, K., Yomagata, H., Sakaguchi, K., and Udaka, S. (1991),J. Bacteriol. 173, 3078–3083.Google Scholar
  16. 16.
    Rangarajan, M. and Hartely, B. S. (1992),Biochem. J. 283, 223–233.Google Scholar
  17. 17.
    Drocourt, D., Bejar, S., Calmels, T., Reynes, J. P., and Tiraby, G. (1988),Nucleic Acids Res. 16, 9337.CrossRefGoogle Scholar
  18. 18.
    Kikuchi, T., Itoh, Y., Kasumi, T., Fukazawa, C. (1990),Agric. Biol. Chem. 54, 2469–2472.Google Scholar
  19. 19.
    Feldmann, S. D., Hremann, S., and Sprenger, G. A. (1992),Mol. Gen. Genet. 234, 201–210.CrossRefGoogle Scholar
  20. 20.
    Bor, -Y. -C, Moraes, C, Lee, -S.-P., Crosby, W. L., Sinsky, A. J., and Batt, C. A. (1992),Gene 114, 127–131.CrossRefGoogle Scholar
  21. 21.
    Chan, -E. -C, Ueng, P. P., and Chen, L. F. (1989),Appl. Microbiol. Biotechnol. 31, 524–528.CrossRefGoogle Scholar
  22. 22.
    Deng, X. X. and Ho, N. W. Y. (1990),Appl. Biochem. Biotechnol. 24/25, 193–199.CrossRefGoogle Scholar
  23. 23.
    Carrel, H. L., Gulsker, J. P., Burger, V., Manfre, F., Tritsch, D. and Biellman, J. P. (1989),Ptroc. Natl. Acad. Sci. USA,86, 4440.CrossRefGoogle Scholar
  24. 24.
    Collyer, C. A., Henrick, K., and Blow, D. M. (1990),J. Mol. Biol. 212, 211–235.CrossRefGoogle Scholar
  25. 25.
    Lehmacher, A. and Biswanger, H. (1990),J. Gen. Microbiol. 136, 679–686.Google Scholar
  26. 26.
    Jensen, V. J. and Rugh, S. (1987),Methods Enzymol. 136, 356–370.Google Scholar
  27. 27.
    Volkin, D. B. and Klibanov, A. M. (1989),Biotechnol. Bioeng. 33, 1104–1111.CrossRefGoogle Scholar
  28. 28.
    Dekker, K., Sugiura, A., Yomagata, H., Sakaguchi, K., and Udaka, S. (1992),Appl. Microbiol. Biotechnol. 36, 727–732.CrossRefGoogle Scholar
  29. 29.
    Merino, E., Balbas, P., Puente, J. L., and Bolivar, F. (1994),Nucleic Acids Res. 22, 1903–1908.CrossRefGoogle Scholar
  30. 30.
    Merino, E., Balbas, P., and Bolivar, F. (1991),Orig. Life. Evol. Biosph. 21, 251–254.CrossRefGoogle Scholar
  31. 31.
    Kumamoto, C. A. and Nault, A. K. (1989),Gene 75, 167–175.CrossRefGoogle Scholar
  32. 32.
    Miller, J. H. (1972), inExperiments in Molecular Genet. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  33. 33.
    Bradford, M. M. (1976),Anal. Biochem. 72, 248–254.CrossRefGoogle Scholar
  34. 34.
    Laemmli, U. K. (1970),Nature 227, 680–685.CrossRefGoogle Scholar
  35. 35.
    Sambrook, J., Fritch, E., and Maniatis, T. (1989), inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  36. 36.
    Hanahan, D. (1983),J. Mol. Biol. 53, 159–162.Google Scholar
  37. 37.
    Bragger, J. M., Daniel, R. M., Coolbear, T., and Morgan, H. W. (1989),Appl. Microbiol. Biotechnol. 31, 556–561.CrossRefGoogle Scholar
  38. 38.
    Novo-Nordisk. (1994), Patent WO9207069.Google Scholar
  39. 39.
    Vieille, C, Hess, J. M., Kelly, R. M., and Zeikus, J. G. (1995),Appl. Environ. Microbiol. 61, 1867–1875.Google Scholar
  40. 40.
    Chen, W.-P. (1980),Process Biochem. Aug–Sep., 30–35.Google Scholar
  41. 41.
    Boyer, H. W. and Roulland-Dussiox, D. (1969),J. Mol. Biol. 41, 459–472.CrossRefGoogle Scholar
  42. 42.
    Amann, E. and Brosius, J. (1985),Gene 40, 183–190.CrossRefGoogle Scholar
  43. 43.
    Oshima, T. and Imahori, K. (1971),J. Gen. Microbiol. 17, 513–517.Google Scholar
  44. 44.
    Vieira, J. and Messing J. (1982),Gene 19, 259–268.CrossRefGoogle Scholar
  45. 45.
    Nash, J. (1991), PrimerGen Program Ver. 1.1, National Research Council of Canada.Google Scholar

Copyright information

© Springer-Verlag 1997

Authors and Affiliations

  • Byoung Chul Park
    • 1
    • 2
  • Sukhoon Koh
    • 1
  • Changsoo Chang
    • 1
    • 3
  • Se Won Suh
    • 1
    • 3
  • Dae-Sil Lee
    • 1
  • Si Myung Byun
    • 2
    • 4
  1. 1.Korea Research Institute of Bioscience and BiotechnologyYusongKorea
  2. 2.Department of Biological ScienceKorea Advanced Institute of Science and TechnologyYusongKorea
  3. 3.Department of ChemistrySeoul National UniversitySeoulKorea
  4. 4.Research Center for New Bio-Materials in AgricultureSeoul National UniversitySuwonKorea

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