Skip to main content
SpringerLink
Log in

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

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

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.

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

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.

Similar content being viewed by others

Abbreviations

BACΔNC:

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

SDS:

sodium dodecyl sulfate

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

TEMED:

N,N,N′,N′-tetramethylethylenediamine

IPTG:

isopropyl-β-d-thiogalactopyranoside

Ni2+-NTA:

nickel nitrilotriacetate

BLA:

Bacillus licheniformis α-amylase

References

  • 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 29:6244–6249

    Article  CAS  Google Scholar 

  • Bradford, M. M. (1976). Anal. Chem. 72:248–254

    CAS  Google Scholar 

  • Buisson G., Duee E., Haser R., and Payan, F. (1987). EMBO J. 6:3909–3916

    CAS  Google Scholar 

  • Chang, C. T., Lo, H. F., Chi, M. C., Yao, C. Y., Hsu, W. H., and Lin, L. L. (2003). Extremophiles 7:505–509

    Article  CAS  Google Scholar 

  • Declerck, N., Machius, M., Wiegand, G., Huber, R., and Gaillardin, C. (2000). J. Mol. Biol. 301:1041–1057

    Article  CAS  Google Scholar 

  • Guzman-Maldonado, H., and Paredes-Lopez, O. (1995). Crit. Rev. Food Sci. Nutr. 35:373–403

    Article  CAS  Google Scholar 

  • Henrissat, B. (1991). Biochem. J. 280:309–316

    CAS  Google Scholar 

  • Ito, S., Kobayashi, T., Ara, K., Ozaki, K., Kawai, S., and Hatada, Y. (1998). Extremophiles 2:185–190

    Article  CAS  Google Scholar 

  • Kang, Y.N., Adachi, M., Utsumi, S., and Mikami, B. (2004). J. Mol. Biol. 339:1129–1140

    Article  CAS  Google Scholar 

  • Karn, R. C. (1978). Adv. Comp. Physiol. Biochem. 7:1–103

    CAS  Google Scholar 

  • Katsuya, Y., Mezaki, Y., Kubota, M., and Matsuura, Y. (1998). J. Mol. Biol. 281:885–897

    Article  CAS  Google Scholar 

  • 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: 26279–26286

  • Kossmann, J., and Lloyd, J. (2000). Crit. Rev. Biochem. Mol. Biol. 35:141–196

    CAS  Google Scholar 

  • Kuriki, T., and Imanaka, T. (1999). J. Biosci. Bioeng. 87:557–565

    Article  CAS  Google Scholar 

  • Laemmli, U. K. (1970). Nature (Lodon) 227:680–685

    Article  CAS  Google Scholar 

  • 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. 236:590–600

    Article  CAS  Google Scholar 

  • Lehmann, M., Pasamontes, L., Lassen, S. F., Wyss, M. (2000). Biochim. Biophys. Acta 1543:408–415

    CAS  Google Scholar 

  • Li, F., and Mullins, J. I. (2002). Methods Mol. Biol. 182:19–27

    CAS  Google Scholar 

  • Lin, L. L., Hsu, W. H., and Chu, W. S. (1997). J. Appl. Microbiol. 82:325–334

    Article  CAS  Google Scholar 

  • Lin, L. L., Tsau, M. R., and Chu, W. S. (1994). Appl. Microbial. Biotechnol. 42:51–56

    Article  CAS  Google Scholar 

  • Lo, H. F., Lin, L. L., Li, C. C., Hsu, W. H., and Chang, C. T. (2001). Curr. Microbiol. 43:170–175

    Article  CAS  Google Scholar 

  • Lo, H. F., Lin, L. L., Chiang, W. Y., Chi, M. C., Hsu, W. H., and Chang, C. T. (2002). Arch. Microbiol. 178:115–123

    Article  CAS  Google Scholar 

  • Machius, M., Wiegand, G., and Huber, R. (1995). J. Mol. Biol. 246:545–559

    Article  CAS  Google Scholar 

  • Matsuura, Y., Kusunoki, M., Harada, W., and Kakudo, M. (1984). J. Biochem (Tokyo) 95:697–702

    CAS  Google Scholar 

  • Miller, G. L. (1959). Anal. Chem. 31:426–428

    Article  CAS  Google Scholar 

  • Morishita, Y., Hasegawa, K., Matsuura, Y., Katsuya, Y., Kubato, M., and Sakai, S. (1997). J. Mol. Biol. 267: 661–672

    Google Scholar 

  • Ohdan, K., Kuriki, T., Kaneko, H., Shimada, J., Takada, T., Fujimoto, Z., Mizuno, H., and Okada, S. (1999). Appl. Environ. Microbiol. 65:4652–4658

    CAS  Google Scholar 

  • Qian, M., Haser, R., and Payan, F. (1993). J. Mol. Biol. 231:758–799

    Article  Google Scholar 

  • Sambrook, J., and Russell, D. W. (2001) In: Molecular Cloning: A Laboratory Manual, Cold spring harbor laboratory Press, New York, pp. 1.31–1.131

  • Sarcabal, P., Remaud-Simeon, M., Willemot, R., Potocki de Montalk, G., Svensson, B., and Monsan, P. (2000). FEBS Lett. 474:33–37

    Article  CAS  Google Scholar 

  • Strokopytov, B., Knegtel, R. M. A., Penninga, D., Roozeboom, H. J., Kalk, K. H., Dijkhuizen, L., and Dijkstra, B. W. (1996). Biochemistry 35:4241–4249

    Article  CAS  Google Scholar 

  • 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. 6:432–436

    Article  CAS  Google Scholar 

  • Vihinen, M., and Mäntsälä, P. (1989). Crit. Rev. Biochem. Mol. Biol. 24:329–418

    Article  CAS  Google Scholar 

  • Watanabe, K., Miyake, K., and Suzuki, Y. (2001). Biosci. Biotechnol. Biochem. 65:2058–2064

    Article  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Department of Applied Chemistry, National Chiayi University, 300 University Road, 60083, Chiayi, Taiwan

    Long-Liu Lin

  2. Department of Food and Nutrition, Hungkuang University, 34 Chungchie Road, Taichung, 433, Taiwan

    Pei-Jing Chen & Huei-Fen Lo

  3. Institute of Molecular and Cellular Biology, and Department of Life Science, National Tsing Hua University, Hsinchu, 300, Taiwan

    Jai-Shin Liu & Wen-Ching Wang

Authors
  1. Long-Liu Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pei-Jing Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jai-Shin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wen-Ching Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Huei-Fen Lo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Huei-Fen Lo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lin, LL., Chen, PJ., Liu, JS. et al. Identification of Glutamate Residues Important for Catalytic Activity or Thermostability of a Truncated Bacillus sp. Strain TS-23 α-amylase by Site-directed Mutagenesis. Protein J 25, 232–239 (2006). https://doi.org/10.1007/s10930-006-9006-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-006-9006-7

Keywords

Search

Navigation