Skip to main content
SpringerLink
Log in

Cloning and expression of thermostable β-glycosidase gene from thermus nonproteolyticus HG102 and characterization of recombinant enzyme

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The gene coding for β-glycosidase (EC 3.2.1.21) from Thermus nonproteolyticus HG102 was cloned and expressed in Escherichia coli. The gene open reading frame was 1311 bp, and it codes for 437 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of the glycosyl hydrolase family I. The enzyme had high content of Arg and Pro. The recombinant enzyme was purified to homogeneity with heat precipitation, ammonium sulfate precipitation, DEAE-cellulose (DE52) chromatography, and prepared slab polyacrylamide gel electrophoresis. The enzyme was monomeric and had a molecular mass of 48,900 Daltons and a pI of 5.2. The enzyme showed optimum activity at pH 5.6 and 90°C. It catalyzed the hydrolysis of β-d-glucoside, β-d-galactoside, β-d-fucoside, and β-d-mannoside. Lineweaver-Burk plots showed that the k cat /K m ratio for β-d-glucoside and β-d-fucoside was higher than for β-d-mannoside and β-d-galactoside. The enzyme was extremely thermostable, with a half-life of 2.5 h at 90°C, and was stable over a wide range of pH. It also had transglycosidic activity at high temperature.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ichikawa, Y., Look, G. C., and Wong, C.-H. (1992), Anal. Biochem. 202, 215–238.

    Article  CAS  Google Scholar 

  2. Thomas, K. N. G. and Kenealy, W. R. (1986), in Thermophiles, Thomas D. Brock. Wiley-Interscience, New York, pp. 197–216.

    Google Scholar 

  3. Kengen, S. W. M., Luesink, E. J., Stams, A. J. M., and Zehnder, A. J. B. (1993), Eur. J. Biochem. 213, 305–312.

    Article  CAS  Google Scholar 

  4. Grogan, D. W. (1991), Appl. Environ. Microbiol. 57, 1644–1649.

    CAS  Google Scholar 

  5. Gabelsberger, J., Liebl W., and Schleifer, K. (1993), FEMS Microbiol. Lett. 109, 131–138.

    CAS  Google Scholar 

  6. Chi, Y.-I., Martinez-Cruz, L. A., Jancarik, J., Swanson, R. V., Robertson, D. E., and Kim, S.-H. (1999), FEBS Lett. 445, 375–383.

    Article  CAS  Google Scholar 

  7. Dion, M., Fourage, L., Hallet, J.-N., and Colas, B. (1999), Glyconcoj. J. 16, 27–37.

    Article  CAS  Google Scholar 

  8. Miaoying, C., Shoujun, Y., Junfeng, L., and Jun, W. (1992), Acta Microbiol. Sinica 32, 233–237.

    Google Scholar 

  9. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  10. Marmur, J. (1961), J. Mol. Biol. 3, 208–218.

    Article  CAS  Google Scholar 

  11. Altschul, S. F., Makken, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997), Nucleic Acids Res. 25(17), 3389–3402.

    Article  CAS  Google Scholar 

  12. Laemmli, U. K. (1970), Nature 227, 680–685.

    Article  CAS  Google Scholar 

  13. Vesterberg, O. (1973), Sci. Tools 20, 22–27.

    CAS  Google Scholar 

  14. Lowry, O. H., Rosebrough, W. J., Farr, A. L., and Randall, R. J. (1951), J. Biol. Chem. 193, 265–275.

    CAS  Google Scholar 

  15. Lineweaver, H. and Burk, D. (1934), J. Am. Chem. Soc. 56, 658–666.

    Article  CAS  Google Scholar 

  16. Perez-Pons, J. A., Cayetano, A., Rebordosa, X., Lloberas, J., Guasch, A., and Queral, E. (1994), Eur. J. Biochem. 223, 557–565.

    Article  CAS  Google Scholar 

  17. Breves, R., Bronnenmeier, K., Wild, N., Lottspeich, R., Standenbaner, W. L., and Hofemeister, J. (1997), Appl. Environ. Microbiol. 63(10), 3902–3910.

    CAS  Google Scholar 

  18. Lieb, W., Gabelsberger, J., and Schleifer, K.-H. (1994), Mol. Gen. Genet. 242, 111–115.

    Google Scholar 

  19. Love, D. R., Fisher, R., and Bergquist, P. L. (1988), Mol. Gen. Genet. 213, 84–92.

    Article  CAS  Google Scholar 

  20. Paavilainen, S., Hellman, J., and Korpela, T. (1993), Appl. Environ. Microbiol. 59(3), 927–932.

    CAS  Google Scholar 

  21. Wright, R. M., Yablonsky, M. D., Shalita, Z. P., Goyal, A. K., and Eveleigh, D. E. (1992), Appl. Environ. Microbiol. 58, 3455–3465.

    CAS  Google Scholar 

  22. Gonzalez-Candelas, L., Ramon, D., and Polaina, J. (1990), Gene 95, 31–38.

    Article  CAS  Google Scholar 

  23. Dakhova, O., Kurepina, N., Zverlov, V., Svetlichnyi, V., and Velikodvorshaya, G. (1993), Biochem. Biophys. Res. Commun. 194, 1359–1364.

    Article  CAS  Google Scholar 

  24. Moracci, M., Capalbo, L., Ciaramella, M., and Rossi, M. (1996), Protein Eng. 9, 1191–1195.

    Article  CAS  Google Scholar 

  25. Chou, P. Y. and Fasman, G. D. (1978), Adv. Enzymol. 47, 45.

    CAS  Google Scholar 

  26. Davies, G. and Henrissat, B. (1995), Structure 33, 853–859.

    Article  Google Scholar 

  27. Aguilar, C. F., Sanderson, I., Moracci, M., Ciaramella, M., Nucci, R., Rossi, M., and Pearl, L. H. (1997), J. Mol. Biol. 271, 789–802.

    Article  CAS  Google Scholar 

  28. Clarke, A. J., Brang, M. R., and Strating, H. (1993), in β-Glucosidase, Esen, A., ed., American Chemical Society, Washington, DC, pp. 27–40.

    Google Scholar 

  29. Folcarelli, S., Battistoni, A., Carri, M. T., Polticelli, F., Falconi, M., Nicolini, L., Stella, L., Rosato, N., Rotilio, G., and Desideri, A. (1996), Protein Eng. 9, 232–235.

    Article  Google Scholar 

  30. Shibuya, H., Abe, M., Sekiguchi, T., and Nosoh, Y. (1980), Biochim. Biophys. Acta 708, 300–304.

    Google Scholar 

  31. Watanabe, K., Kitamura, K., and Suzuki, Y. (1996), Appl. Environ. Microbiol. 62, 2066–2073.

    CAS  Google Scholar 

  32. Schimmel, P. R. and Flory, P. J. (1968), J. Mol. Biol. 34, 105–120.

    Article  CAS  Google Scholar 

  33. Levitt, M. (1978), Biochemistry 17, 4277–4284.

    Article  CAS  Google Scholar 

  34. Suzuki, Y., Oishi, K., Nakano, H., and Nagayama, T. (1987), Appl. Microbiol. Biotechnol. 26, 546–551.

    Article  CAS  Google Scholar 

  35. Vieille, C. and Zeikus, J. G. (1996), Trends Biotechnol. 14, 183–190.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Microbiology, Chinese Academy of Science, 100080, Beijing, China

    He Xiangyuan, Zhang Shuzheng & Yang Shoujun

Authors
  1. He Xiangyuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhang Shuzheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yang Shoujun
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

He, X., Zhang, S. & Yang, S. Cloning and expression of thermostable β-glycosidase gene from thermus nonproteolyticus HG102 and characterization of recombinant enzyme. Appl Biochem Biotechnol 94, 243–255 (2001). https://doi.org/10.1385/ABAB:94:3:243

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1385/ABAB:94:3:243

Index Entries

Search

Navigation