The Protein Journal

, 25:232 | Cite as

Identification of Glutamate Residues Important for Catalytic Activity or Thermostability of a Truncated Bacillus sp. Strain TS-23 α-amylase by Site-directed Mutagenesis

  • Long-Liu Lin
  • Pei-Jing Chen
  • Jai-Shin Liu
  • Wen-Ching Wang
  • Huei-Fen Lo


The importance of 17 glutamate residues of a truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was investigated by site-directed mutagenesis. The Ala- and Asp-substituted variants were overexpressed in the recombinant E. coli cells and the 54-kDa proteins were purified to nearly homologous by nickel-chelate chromatography. Glu-295, which locates in the conserved region III of amylolytic enzymes, mutations resulted in a complete loss of enzyme activity. The specific activity for E151A was decreased by more than 30%, while other variants showed activity comparable to that of BACΔNC. A decreased half-life at 70°C was observed for Glu-219 variants with respective to the wild-type enzyme, suggesting that replacement of Glu-219 by either Ala or Asp might have a significant destabilizing effect on the protein structure.


α-Amylase Bacillus sp. strain TS-23 catalytic reaction site-directed mutagenesis thermostability 



the N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase


sodium dodecyl sulfate


SDS-polyacrylamide gel electrophoresis






nickel nitrilotriacetate


Bacillus licheniformis α-amylase



This work was supported by Grants NSC 93-2313-B-241-005 and NSC 94-2313-B-241-005 from National Science Council of Taiwan, Republic of China.


  1. Boel E., Brady L., Brzozowski A. M., Derewenda Z., Dodson G. G., Jensen V. J., Petersen S. B., Swift H., Thim L., Woldike H. F. (1990). Biochemistry 29:6244–6249CrossRefGoogle Scholar
  2. Bradford, M. M. (1976). Anal. Chem. 72:248–254Google Scholar
  3. Buisson G., Duee E., Haser R., and Payan, F. (1987). EMBO J. 6:3909–3916Google Scholar
  4. Chang, C. T., Lo, H. F., Chi, M. C., Yao, C. Y., Hsu, W. H., and Lin, L. L. (2003). Extremophiles 7:505–509CrossRefGoogle Scholar
  5. Declerck, N., Machius, M., Wiegand, G., Huber, R., and Gaillardin, C. (2000). J. Mol. Biol. 301:1041–1057CrossRefGoogle Scholar
  6. Guzman-Maldonado, H., and Paredes-Lopez, O. (1995). Crit. Rev. Food Sci. Nutr. 35:373–403CrossRefGoogle Scholar
  7. Henrissat, B. (1991). Biochem. J. 280:309–316Google Scholar
  8. Ito, S., Kobayashi, T., Ara, K., Ozaki, K., Kawai, S., and Hatada, Y. (1998). Extremophiles 2:185–190CrossRefGoogle Scholar
  9. Kang, Y.N., Adachi, M., Utsumi, S., and Mikami, B. (2004). J. Mol. Biol. 339:1129–1140CrossRefGoogle Scholar
  10. Karn, R. C. (1978). Adv. Comp. Physiol. Biochem. 7:1–103Google Scholar
  11. Katsuya, Y., Mezaki, Y., Kubota, M., and Matsuura, Y. (1998). J. Mol. Biol. 281:885–897CrossRefGoogle Scholar
  12. Kim, J. S., Chan, S. S., Kim, H. J., Kim, T. J., Ha, N. C., Oh, S. T., Cho, H. S., Cho, M. J., Kim, M. J., Lee, H. S., Kim, J. W., Choi, K. Y., Park, K. H., and Oh, B. H. (1999). J. Biol. Chem. 274: 26279–26286Google Scholar
  13. Kossmann, J., and Lloyd, J. (2000). Crit. Rev. Biochem. Mol. Biol. 35:141–196Google Scholar
  14. Kuriki, T., and Imanaka, T. (1999). J. Biosci. Bioeng. 87:557–565CrossRefGoogle Scholar
  15. Laemmli, U. K. (1970). Nature (Lodon) 227:680–685CrossRefGoogle Scholar
  16. Lawson, C. L., van Montfort, R., Strokopytov, B., Rozeboom, H. J., Kalk, K. H., de Vries, G., Penninga, D., Dijkhuizen, L., and Dijkstra, B. W. (1994). J. Mol. Biol. 236:590–600CrossRefGoogle Scholar
  17. Lehmann, M., Pasamontes, L., Lassen, S. F., Wyss, M. (2000). Biochim. Biophys. Acta 1543:408–415Google Scholar
  18. Li, F., and Mullins, J. I. (2002). Methods Mol. Biol. 182:19–27Google Scholar
  19. Lin, L. L., Hsu, W. H., and Chu, W. S. (1997). J. Appl. Microbiol. 82:325–334CrossRefGoogle Scholar
  20. Lin, L. L., Tsau, M. R., and Chu, W. S. (1994). Appl. Microbial. Biotechnol. 42:51–56CrossRefGoogle Scholar
  21. Lo, H. F., Lin, L. L., Li, C. C., Hsu, W. H., and Chang, C. T. (2001). Curr. Microbiol. 43:170–175CrossRefGoogle Scholar
  22. Lo, H. F., Lin, L. L., Chiang, W. Y., Chi, M. C., Hsu, W. H., and Chang, C. T. (2002). Arch. Microbiol. 178:115–123CrossRefGoogle Scholar
  23. Machius, M., Wiegand, G., and Huber, R. (1995). J. Mol. Biol. 246:545–559CrossRefGoogle Scholar
  24. Matsuura, Y., Kusunoki, M., Harada, W., and Kakudo, M. (1984). J. Biochem (Tokyo) 95:697–702Google Scholar
  25. Miller, G. L. (1959). Anal. Chem. 31:426–428CrossRefGoogle Scholar
  26. Morishita, Y., Hasegawa, K., Matsuura, Y., Katsuya, Y., Kubato, M., and Sakai, S. (1997). J. Mol. Biol. 267: 661–672Google Scholar
  27. Ohdan, K., Kuriki, T., Kaneko, H., Shimada, J., Takada, T., Fujimoto, Z., Mizuno, H., and Okada, S. (1999). Appl. Environ. Microbiol. 65:4652–4658Google Scholar
  28. Qian, M., Haser, R., and Payan, F. (1993). J. Mol. Biol. 231:758–799CrossRefGoogle Scholar
  29. Sambrook, J., and Russell, D. W. (2001) In: Molecular Cloning: A Laboratory Manual, Cold spring harbor laboratory Press, New York, pp. 1.31–1.131Google Scholar
  30. Sarcabal, P., Remaud-Simeon, M., Willemot, R., Potocki de Montalk, G., Svensson, B., and Monsan, P. (2000). FEBS Lett. 474:33–37CrossRefGoogle Scholar
  31. Strokopytov, B., Knegtel, R. M. A., Penninga, D., Roozeboom, H. J., Kalk, K. H., Dijkhuizen, L., and Dijkstra, B. W. (1996). Biochemistry 35:4241–4249CrossRefGoogle Scholar
  32. Uitdehaag, J. C. M., Mosi, R., Kalk, K. H., van der Veen, B. A., Dijkhuizen, L., Withers, S. G., and Dijkstra, B. W. (1999). Nat. Struct. Biol. 6:432–436CrossRefGoogle Scholar
  33. Vihinen, M., and Mäntsälä, P. (1989). Crit. Rev. Biochem. Mol. Biol. 24:329–418CrossRefGoogle Scholar
  34. Watanabe, K., Miyake, K., and Suzuki, Y. (2001). Biosci. Biotechnol. Biochem. 65:2058–2064CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Long-Liu Lin
    • 1
  • Pei-Jing Chen
    • 2
  • Jai-Shin Liu
    • 3
  • Wen-Ching Wang
    • 3
  • Huei-Fen Lo
    • 2
  1. 1.Department of Applied ChemistryNational Chiayi UniversityChiayiTaiwan
  2. 2.Department of Food and NutritionHungkuang UniversityTaichungTaiwan
  3. 3.Institute of Molecular and Cellular Biology, and Department of Life ScienceNational Tsing Hua UniversityHsinchuTaiwan

Personalised recommendations