Skip to main content
Springer Nature Link
Log in

Role of the Calcium-Binding Residues Asp231, Asp233, and Asp438 in Alpha-Amylase of Bacillus amyloliquefaciens as Revealed by Mutational Analysis

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Role of the calcium-binding residues Asp231, Asp233, and Asp438 of Bacillus amyloliquefaciens α-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233, and Asp438 were replaced with Asn, Asn, and Gly to produce the mutants D231N, D233N, and D438G, respectively. The mutant amylases were purified to homogeneity and the purified enzymes was estimated to be approximately 58 kDa. The specific activity for the mutant enzyme D233N was decreased by 84.8%, while D231N and D438G showed a decrease of 6.3% and 3.5% to that of the wild-type enzyme, respectively. No significant changes in the K m value, thermo-stability, optimum temperature, and optimum pH were observed in the mutations of D231N and D438G, while substitution of Asp233 with Asn resulted in a dramatic reduction in the value of catalytic efficiency (K cat/K m) and thermo-stability at 60°C. The ranges of optimum temperature and optimum pH for D233N were also reduced to about 10°C and 3–4 units, respectively.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  1. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R (2000) Advances in microbial amylases. Biotechnol Appl Biochem 31(Pt 2):135–152

    Article  CAS  PubMed  Google Scholar 

  2. Pretorius IS, Laing E, Pretorius GH, Marmur J (1988) Expression of a Bacillus alpha-amylase gene in yeast. Curr Genet 14:1–8

    Article  CAS  PubMed  Google Scholar 

  3. Cherry JR, Fidantsef AL (2003) Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol 14:438–443

    Article  CAS  PubMed  Google Scholar 

  4. Nielsen JE, Borchert TV (2000) Protein engineering of bacterial alpha-amylases. Biochim Biophys Acta 1543:253–274

    CAS  PubMed  Google Scholar 

  5. Janecek S (1997) alpha-Amylase family: molecular biology and evolution. Prog Biophys Mol Biol 67:67–97

    Article  CAS  PubMed  Google Scholar 

  6. Kuriki T, Imanaka T (1999) The concept of the alpha-amylase family: structural similarity and common catalytic mechanism. J Biosci Bioeng 87:557–565

    Article  CAS  PubMed  Google Scholar 

  7. Tanaka A, Hoshino E (2003) Secondary calcium-binding parameter of Bacillus amyloliquefaciens alpha-amylase obtained from inhibition kinetics. J Biosci Bioeng 96:262–267

    CAS  PubMed  Google Scholar 

  8. Brzozowski AM, Lawson DM, Turkenburg JP, Bisgaard-Frantzen H, Svendsen A, Borchert TV, Dauter Z, Wilson KS, Davies GJ (2000) Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes. Biochemistry 39:9099–9107

    Article  CAS  PubMed  Google Scholar 

  9. Lee S, Mouri Y, Minoda M, Oneda H, Inouye K (2006) Comparison of the wild-type alpha-amylase and its variant enzymes in Bacillus amyloliquefaciens in activity and thermal stability, and insights into engineering the thermal stability of Bacillus alpha-amylase. J Biochem 139:1007–1015

    Article  CAS  PubMed  Google Scholar 

  10. Voskuil MI, Chambliss GH (1993) Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis. Appl Environ Microbiol 59:1138–1142

    CAS  PubMed  Google Scholar 

  11. Gryczan TJ, Dubnau D (1978) Construction and properties of chimeric plasmids in Bacillus subtilis. Proc Natl Acad Sci USA 75:1428–1432

    Article  CAS  PubMed  Google Scholar 

  12. Dieffenbach CW, Dveksler GS (2004) PCR primer: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  13. Zhi WB, Deng QY, Song JG, Gu M, Ouyang F (2005) One-step purification of alpha-amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography with aqueous polymer two-phase systems. J Chromatogr A 1070:215–219

    Article  CAS  PubMed  Google Scholar 

  14. Laemmli U (1970) Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  15. Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  16. Xiao ZZ, Storms R, Tsang A (2006) A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Anal Biochem 351:146–148

    Article  CAS  PubMed  Google Scholar 

  17. Palva I (1982) Molecular cloning of alpha-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis. Gene 19:81–87

    Article  CAS  PubMed  Google Scholar 

  18. Priyadharshini R, Gunasekaran P (2007) Site-directed mutagenesis of the calcium-binding site of alpha-amylase of Bacillus licheniformis. Biotechnol Lett 29:1493–1499

    Article  CAS  PubMed  Google Scholar 

  19. Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C (2000) Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. J Mol Biol 301:1041–1057

    Article  CAS  PubMed  Google Scholar 

  20. Savchenko A, Vieille C, Kang S, Zeikus JG (2002) Pyrococcus furiosus alpha-amylase is stabilized by calcium and zinc. Biochemistry 41:6193–6201

    Article  CAS  PubMed  Google Scholar 

  21. Lim JK, Lee HS, Kim YJ, Bae SS, Jeon JH, Kang SG, Lee JH (2007) Critical factors to high thermostability of an alpha-amylase from hyperthermophilic archaeon Thermococcus onnurineus NA1. J Microbiol Biotechnol 17:1242–1248

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the grants from the National High Technology Research and Development Program of China (863 Program) (No. 2006AA020204 and No. 2006AA10Z307). The authors thank Bacillus Genetic Stock Center for kindly providing the strains and vectors.

Author information

Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Yang Liu, Wei Shen, Gui-yang Shi & Zheng-xiang Wang

  2. Center for Bioresource & Bioenergy, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Yang Liu, Wei Shen, Gui-yang Shi & Zheng-xiang Wang

Authors
  1. Yang Liu
    View author publications

    Search author on:PubMed Google Scholar

  2. Wei Shen
    View author publications

    Search author on:PubMed Google Scholar

  3. Gui-yang Shi
    View author publications

    Search author on:PubMed Google Scholar

  4. Zheng-xiang Wang
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Zheng-xiang Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, Y., Shen, W., Shi, Gy. et al. Role of the Calcium-Binding Residues Asp231, Asp233, and Asp438 in Alpha-Amylase of Bacillus amyloliquefaciens as Revealed by Mutational Analysis. Curr Microbiol 60, 162–166 (2010). https://doi.org/10.1007/s00284-009-9517-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-009-9517-5

Keywords