The Protein Journal

, Volume 24, Issue 2, pp 79–88 | Cite as

Effect of Glycosylation on the Catalytic and Conformational Stability of Homologous α-Amylases



A thermostable α-amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca2+. The stability of the native enzyme was found to increase upon modification as observed from the increase in t1/2 in the absence of Ca2+ ions by about 1.5–13.7 times (at 85°C) in the case of BLA and 5.7–8.4 times (at 50°C) for BAA. The highest stability was observed for OSP400 modified enzyme with ΔCm and ΔTm values of 0.63 M and 7.92°C for BLA and 0.85 M and 5.3°C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.


α-Amylase conformational stability covalent modification stabilization sucrose polymer unfolding 


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  1. Cavicchioli, R., Siddiqui, K.S., Andrews, D., Sowers, K.R. 2002Curr. Opin. Biotechnol.13253261CrossRefPubMedGoogle Scholar
  2. Coleman, G., Elliott, W.H. 1962Biochem. J.83256263PubMedGoogle Scholar
  3. Conrad, B., Hoang, V., Polley, A., Hofemeister, J. 1995Eur. J. Biochem.230481490PubMedGoogle Scholar
  4. D’Amico, S., Marx, J.-C., Gerday, C., Feller, G. 2003J.␣Biol. Chem.27878917896CrossRefPubMedGoogle Scholar
  5. De Cordt, S., Hendrickx, M., Maesmans, G., Tobback, P. 1994Biotechnol. Bioengng.43107114CrossRefGoogle Scholar
  6. Declerck, N., Joyet, P., Trosset, J.Y., Garnier, J., Gaillardin, C. 1995Protein. Engng.810291037Google Scholar
  7. Declerck, N., Machius, M., Chambert, R., Wiegand, G., Huber, R., Gaillardin, C. 1997Protein. Engng.10541549CrossRefGoogle Scholar
  8. Declerck, N., Machius, M., Wiegand, G., Huber, R., Gaillardin, C. 2000J. Mol. Biol.30110411057CrossRefPubMedGoogle Scholar
  9. Declerk, N., Machius, M., Joyet, P., Wiegand, G., Huber, R., Gaillardin, C. 2003Protein Eng.16287293CrossRefPubMedGoogle Scholar
  10. Feller, G., d’Amico, D., Gerday, C. 1999Biochemistry3846134619CrossRefPubMedGoogle Scholar
  11. Feller, G., Gerday, C. 1997Cell Mol. Life Sci.53830841CrossRefPubMedGoogle Scholar
  12. Feller, G., Payan, F., Theys, F., Qian, M., Haser, R., Gerday, C. 1994Eur. J. Biochem.222441447CrossRefPubMedGoogle Scholar
  13. Fitter, J., Herrman, R., Dencher, N.A., Blume, A., Hauss, T. 2001Biochemistry401072310731CrossRefPubMedGoogle Scholar
  14. Giver, L., Gershenson, A., Fresskgard, P.-O., Arnold, F.H. 1998Proc. Natl. Acad. Sci. USA951280912813CrossRefPubMedGoogle Scholar
  15. Gómez, L., Ramírez, H.L., Villalonga, R. 2001Acta. Biotechnol.21265273CrossRefGoogle Scholar
  16. Hernaiz, M.J., Sanchez-Montero, J.M., Sinisterra, J.V. 1996Biotechnol. Tech.10917922Google Scholar
  17. Jaenicke, R., Böhm, G. 1998Curr. Opin. Struct. Biol.8738748CrossRefPubMedGoogle Scholar
  18. Janecek, S., Baláž, S. 1992FEBS Lett.30413CrossRefPubMedGoogle Scholar
  19. Khajeh, K., Gorgani, N.-M. 2001Appl. Biochem.Biotechnol.904755CrossRefPubMedGoogle Scholar
  20. Lee, J.C., Timasheff, S.N. 1981J. Biol. Chem.25671937201PubMedGoogle Scholar
  21. Lis, H., Sharon, N. 1993Eur. J. Biochem.218127CrossRefPubMedGoogle Scholar
  22. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. 1951J. Biol. Chem.193265275PubMedGoogle Scholar
  23. Machius, M., Wiegand, G., Huber, R. 1995J. Mol. Biol.246545559CrossRefPubMedGoogle Scholar
  24. Machius, M., Vèrlèsy, L., Huber, R., Wiegand, G. 1996J. Mol. Biol.260409421CrossRefPubMedGoogle Scholar
  25. Marshall, J.J. 1978Trends. Biochem. Sci.37983CrossRefGoogle Scholar
  26. Pedrosa, C., Fernanda, G., Felice, D., Trisciuzzi, C., Ferreira, S.T. 2000Arch. Biochem. Biophys.382203210CrossRefPubMedGoogle Scholar
  27. Rajalakshmi, N., Sundaram, P.V. 1995Protein Engng.810391047Google Scholar
  28. Stocks, S.J., Jones, A.J.M., Ramey, C.W., Brooks, D.E. 1986Anal. Biochem.154232234CrossRefPubMedGoogle Scholar
  29. Sundaram, P. V., and Srimathi, S.(2004). In: Svendsen, A. (ed.), Enzyme Functionality Design Engineering and Screening. Marcel Dekker, NY, pp. 632–661.Google Scholar
  30. Sundaram, P.V., Venkatesh, R. 1998Protein Engng.11699705CrossRefGoogle Scholar
  31. Tendian, S.W., Myszka, D.G., Sweet, R.W., Chaiken, I.M., Brouillette, C.G. 1995Biochemistry3464646474CrossRefPubMedGoogle Scholar
  32. Terashima, M., and Katoh, S. (1996). In: Dordick, J. S., and Russell A. J. (ed.), Enzyme Engineering XIII. Ann. N.Y. Acad. Sci. 799: 65–69.Google Scholar
  33. Tomazic, S.J., Klibanov, A.M. 1988J. Biol. Chem.26330863091PubMedGoogle Scholar
  34. Tor, R., Dror, Y., Freeman, A. 1989Enzyme Microb.Technol.11306312CrossRefGoogle Scholar
  35. Venkatesh, R., Sundaram, P.V. 1998aProtein Engng.11691698CrossRefGoogle Scholar
  36. Venkatesh, R., Sundaram, P.V. 1998Ann. N.Y. Acad. Sci.864517520Enzyme Engineering XIVGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  1. 1.Centre for Protein Engineering and Biomedical ResearchThe Voluntary Health ServicesChennaiIndia

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