Biotechnology Letters

, Volume 29, Issue 10, pp 1493–1499 | Cite as

Site-directed mutagenesis of the calcium-binding site of α-amylase of Bacillus licheniformis

  • Ramachandran Priyadharshini
  • Paramasamy Gunasekaran
Original Research Paper
First Online:
Received:
Accepted:

Abstract

Amylases that are active under acidic conditions (pH <6), at higher temperatures (>70°C) and have less reliance on Ca2+ are required for starch hydrolysis. The α-amylase gene of Bacillus licheniformis MTCC 6598 was cloned and expressed in Escherichia coli BL21. The calcium-binding site spanning amino acid residues from 104 to 200 in the loop regions of domain B and D430 in domain C of amylase were changed by site-directed mutagenesis and the resultant mutant amylases were analyzed. Calcium-binding residues, N104, D161, D183, D200 and D430, were replaced with D104 and N161, N183, N200 and N430, respectively. Mutant amylase with N104D had a slightly decreased activity at 30°C but a significantly improved specific activity at pH 5 and 70°C, which is desirable character for a food enzyme. The amylase mutants with D183N or D200N lost all activity while the mutant amylase with D161N retained its activity at 30°C but had significantly less activity at 70°C. On the other hand, the activity of the mutant amylase with D430N was not changed at 30°C but had an improved activity at 70°C.

Keywords

α-Amylases Bacillus licheniformis Calcium binding Site-directed mutagenesis 
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Notes

Acknowledgements

The authors thank Department of Biotechnology, India for the financial assistant through project No BT/PR 4549/PID/20/176/2003 and Center for Excellence in Genomics, Madurai Kamaraj University for providing the infrastructure facilities.

References

  1. Brzozowski AM, Lawson DM, Turkenburg JP, Frantzen HB, 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 39:9099–9107PubMedCrossRefGoogle Scholar
  2. DeLano WL (2003) PyMOL reference manual. DeLano Scientific LLC, San Carlos, CAGoogle Scholar
  3. Declerck N, Joyet P, Trosset JY, Garnier J, Gaillardin C (1995) Hyperthermostable mutants of Bacillus licheniformis α-amylase: multiple amino acid replacements and molecular modelling. Protein Eng 8:1029–1037PubMedCrossRefGoogle Scholar
  4. Declerck N, Machius M, Chambert R, Wiegand G, Huber R, Gaillardin C (1997) Hyperthermostable mutants of Bacillus licheniformis α-amylase: thermodynamic studies and structural interpretation. Protein Eng 10:541–549PubMedCrossRefGoogle Scholar
  5. Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C (2000) Probing structural determinants specifying high thermostability in Bacillus licheniformis α-Amylase. J Mol Biol 301:1041–1057PubMedCrossRefGoogle Scholar
  6. Declerck N, Machius M, Joyet P, Wiegand G, Huber R, Gaillardin C (2002) Engineering the thermostability of Bacillus licheniformis α-amylase. Biologia Bratislava 11:203–211Google Scholar
  7. Declerck N, Machius M, Joyet P, Wiegand G, Huber R, Gaillardin C (2003) Hyperthermostabilization of Bacillus licheniformis α-amylase and modulation of its stability over a 50°C temperature range. Protein Eng 16:101–107CrossRefGoogle Scholar
  8. Hagihara H, Hayashi Y, Endo K, Igarashi K, Ozawa T, Kawai S, Ozaki K, Ito S (2001) Deduced amino-acid sequence of a calcium-free alpha-amylase from a strain of Bacillus: implications from molecular modeling of high oxidation stability and chelator resistance of the enzyme. Eur J Biochem 268:3974–3982PubMedCrossRefGoogle Scholar
  9. Hashida M, Bisgaard-Frantzen H (2000) Protein engineering of new industrial amylases. Trends Glycosci Glycotechnol 12:389–401Google Scholar
  10. Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  11. Igarashi K, Hatada Y, Ikawa K, Araki H, Ozawa T, Kobayashi T, Ozaki K, Ito S (1998a) Improved thermostability of a Bacillus α-amylase by deletion of an arginine-glycine residue is caused by enhanced calcium binding. Biochem Biophys Res Commun 248:372–377PubMedCrossRefGoogle Scholar
  12. Igrashi K, Hatada Y, Hagihara H, Saeki K, Takaiwa M, Uemura T, Ara K, Ozaki K, Kawai S, Kobayashi T et al (1998b) Enzymatic properties of a novel liquefying α amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences .Appl Environ Microbiol 64:3282–3289Google Scholar
  13. Janecek S (1997) α-Amylase family: molecular biology and evolution. Prog Biophys Mol Biol 67:67–97PubMedCrossRefGoogle Scholar
  14. Kuriki T, Kaneko H, Yanase M, Takata H, Shimada J, Handa S, Takada T, Umeyama H, Okada S (1996) Controlling substrate preference and transglycosylation activity of neopullulanase by manipulating steric constraint and hydrophobicity in active center. J Biol Chem 271:17321–17329PubMedCrossRefGoogle Scholar
  15. Lin LL, Chyau CC, Hsu WH (1998) Production and properties of a raw-starch-degrading α-amylase from thermophilic and alkaliphilic Bacillus sp. TS-23 Biotech Appl Biochem 28:61–68Google Scholar
  16. Machius M, Declerck N, Huber R, Wiegand G (1998) Activation of Bacillus licheniformis α-amylase through a disorder–order transition of the substrate-binding site mediated by a calcium–sodium–calcium metal triad. Structure 6:281–292PubMedCrossRefGoogle Scholar
  17. Matsui I, Svensson B (1997) Improved activity and modulated action pattern obtained by random mutagenesis at the fourth beta-alpha loop involved in substrate binding to the catalytic (β/α)8–barrel domain of barley α-amylase. J Biol Chem 272:22456–22463PubMedCrossRefGoogle Scholar
  18. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor., NYGoogle Scholar
  19. Nakajima R, Imanaka T, Aiba S (1985) Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene. J Bacteriol 163:401–406PubMedGoogle Scholar
  20. Nonaka T, Fujihashi M, Kita A, Hagihara H, Ozaki K, Ito S, Miki K (2003) Crystal structure of calcium-free alpha-amylase from Bacillus sp. strain KSM-K38 (AmyK38) and its sodium ion binding sites. J Biol Chem 278:24818–24824PubMedCrossRefGoogle Scholar
  21. Nielsen JE, Beier L, Otzen D, Borchert TV, Frantzen HB, Andersen KV, Svendsen A (1999) Electrostatics in the active site of an α-amylase. Eur J Biochem 264: 816–824PubMedCrossRefGoogle Scholar
  22. Shaw A, Bott R, Day AG (1999) Protein engineering of α-amylase for low pH performance. Curr Opin Biotechnol 10:349–352PubMedCrossRefGoogle Scholar
  23. Suvd D, Fujimoto Z, Takase K, Matsumura M, Mizuno H (2001) Crystal structure of Bacillus stearothermophilus alpha-amylase: possible factors determining the thermostability. J Biochem 129:461–468PubMedGoogle Scholar
  24. Takkinen K, Pettersson RF, Kalkkinen N, Palva I, Soederlund H, Kaeaeriaeinen L (1983) Amino acid sequence of alpha-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene. J Biol Chem 258:1007–1013PubMedGoogle Scholar
  25. Yuuki T, Nomura T, Tezuka H, Tsuboi A, Yamagata H, Tsukagoshi N, Udaka S (1985) complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences. J Biochem 98:1147–1156PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Ramachandran Priyadharshini
    • 1
  • Paramasamy Gunasekaran
    • 1
  1. 1.Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological SciencesMadurai Kamaraj UniversityMaduraiIndia

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