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http://dx.doi.org/10.4014/jmb.1008.08013

Repeated Random Mutagenesis of ${\alpha}$-Amylase from Bacillus licheniformis for Improved pH Performance  

Priyadharshini, Ramachandran (Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University)
Manoharan, Shankar (Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University)
Hemalatha, Devaraj (Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University)
Gunasekaran, Paramasamy (Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University)
Publication Information
Journal of Microbiology and Biotechnology / v.20, no.12, 2010 , pp. 1696-1701 More about this Journal
Abstract
The ${\alpha}$-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis ${\alpha}$-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.
Keywords
Directed evolution; high-throughput screening; error-prone PCR; Bacillus ${\alpha}$-amylase; random mutagenesis;
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1 Arnold, F. H. and A. A. Volkov. 1999. Directed evolution of biocatalysts. Curr. Opin. Chemi Biol. 3: 54-59.   DOI   ScienceOn
2 Suzuki, Y., N. Ito, T. Yuuki, H. Yamagata, and S. Udaka. 1989. Amino acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation. J. Biol. Chem. 15: 18933-18938.
3 Van der Maarel, M. J. E. C, B. van der Veen, J. C. M. Uitdehaag, H. Leemhuis, and L. Dijkhuizen. 2002. Properties and applications of starch converting enzymes of the $\alpha$-amylase family. J. Biotechnol. 94: 137-155.   DOI   ScienceOn
4 Voigt, C. A., S. L. Mayo, F. H. Arnold, and Z. G. Wang. 2001. Computational method to reduce the search space for directed protein evolution. Proc. Natl. Acad. Sci. U.S.A. 98: 3778-3783.   DOI   ScienceOn
5 Wong, D. W. S., S. B. Batt, C. C. Lee, and G. H. Robertson. 1999. High-activity barley $\alpha$-amylase by directed evolution. Prot. J. 23: 453-460.
6 Lee, S., Y. Mouri, M. Minoda, H. Oneda, and K. Inouye. 2006. Comparison of the wild-type-amylase and its variant enzymes in Bacillus amyloliquefaciens in activity and thermal stability, and insights into engineering the thermal stability of Bacillusamylase. J. Biochem. 139: 1007-1015.   DOI   ScienceOn
7 Miller, L. H. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 246-248.
8 Nielsen, J., E. Torben, V. Borchert, and V. Gerrit. 2001. The determinants of $\alpha$-amylase pH-activity profiles. Prot. Eng. 14: 505-512.   DOI
9 Priyadharshini, R. and P. Gunasekaran. 2007. Site-directed mutagenesis of the calcium binding site of $\alpha$-amylase of Bacillus licheniformis. Biotech. Lett. 29: 1493-1499.   DOI   ScienceOn
10 Richardson, T. H., T. Xuqiu, F. Gerhard, C. Walter, C. Mark, L. David, et al. 2002. A novel, high performance enzyme for starch liquefaction. J. Biol. Chem. 277: 26501-26507.   DOI   ScienceOn
11 Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
12 Schafer, K., U. Magnusson, F. Scheffel, A. Schiefner, M. O. Sandgren, K. Diederichs, et al. 2004. X-Ray structures of the maltose-maltodextrin-binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins. J. Mol. Biol. 335: 261-274.   DOI   ScienceOn
13 Schwede, T., J. Kopp, N. Guex, and M. C. Peitsch. 2003. SWISS-MODEL - An automated protein homology-modeling server. Nucl. Acids Res. 31: 3381-3385.   DOI   ScienceOn
14 Joyet, P., N. Declerck, and C. Gaillardin. 1992. Hyperthermostable variants of a highly thermostable $\alpha$-amylase. Biotechnology 10: 1579-1583.   DOI   ScienceOn
15 Shankar, M., R. Priyadharshini, and P. Gunasekaran. 2009. Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries. Biotech. Lett. 31: 1197-1201.   DOI   ScienceOn
16 Sheryl, B., P. Rubin, and H. Zhao. 2006. Recent advances in biocatalysis by directed enzyme evolution. Combin. Chem. High Through. Screen. 7: 480-485.
17 Jones, A., M. Lamsa, T. P. Frandsen, T. Spendler, P. Harris, A. Sloma, F. Xu, J. B. Nielsen, and J. R. Cherry. 2008. Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25. J. Biotechnol. 134: 325-333.   DOI   ScienceOn
18 Keohavong, P. and W. G. Thilly. 1989. Fidelity of DNA polymerases in DNA amplification assay: Denaturing gradient gel electrophoresis. Proc. Natl. Acad. Sci. U.S.A. 86: 9253-9257.   DOI   ScienceOn
19 Emond, S., G. Potocki-Veronese, P. Mondon, K. Bouayadi, H. Kharrat, P. Monsan, and M. Remaud-Simeon. 2007. Optimized and automated protocols for high-throughput screening of amylosucrase libraries. J. Biomol. Screen. 12: 715-723.   DOI   ScienceOn
20 Kim, Y. W., J. H. Choi, J. W. Kim, C. Park, J. W. Kim, H. Cha, et al. 2003. Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. Appl. Environ. Microbiol. 69: 4866-4874.   DOI   ScienceOn
21 Fushinobu, S., K. Ito, M. Konno, T. Wakagi, and H. Matsuzawa. 1998. Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: Biased distribution of acidic residues and importance of Asp37 for catalysis at low pH. Prot. Eng. 11: 1121-1128.   DOI
22 DeLano, W. L. 2003. PyMOL Reference Manual. DeLano Scientific LLC, San Carlos, CA.
23 Hagihara, H., K. Igarashi, Y. Hayashi, K. Endo, K. Ikawa-Kitayama, K .Ozaki, S. Kawai, and S. Ito. 2001. Novel alpha-amylase that is highly resistant to chelating reagents and chemical oxidants from the alkaliphilic Bacillus isolate KSM-K38. Appl. Environ. Microbiol. 67: 1744-1750.   DOI   ScienceOn
24 Igarashi, K., Y. Hatada, K. Ikawa, H. Araki, T. Ozawa, T. Kobayashi, K. Ozaki, and S. Ito. 1998. Improved thermostability of a Bacillus $\alpha$-amylase by deletion of an arginine-glycine residue is caused by enhanced calcium binding. Biochem. Biophys. Res. Commun. 248: 372-377.   DOI   ScienceOn
25 Conrad, B., V. Hoang, A. Polley, and J. Hofemeister. 1995. Hybrid Bacillus amyloliquefaciens ${\times}$ Bacillus licheniformis alpha-amylases. Construction, properties and sequence determinants. Eur. J. Biochem. 230: 481-490.
26 Demirjian, D. C., F. Moris-Varas, and C. S. Cassidy. 2001. Enzymes from extremophiles. Curr. Opin. Chem. Biol. 5: 144-151.   DOI   ScienceOn
27 Cadwell, R. C. and G. F. Joyce.1994. Mutagenic PCR. PCR Methods Appl. 3: 136-140.   DOI
28 Arnold, K. L., J. K. Bordoli, and T. Schwede. 2006. The SWISS-MODEL Workspace: A Web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201.   DOI   ScienceOn
29 Bessler, C., J. Schmitt, K. H. Maurer, and R. Schmid. 2003. Directed evolution of a bacterial $\alpha$-amylase: Towards enhanced pH-performance and higher specific activity. Prot. Sci. 12: 2141-2149.
30 Bisgaard-Frantzen, H., A. Svendsen, B. Norman, S. Pedersen, S. Kjærulff, H. Outtrup, and T. V. Borchert. 1999. Development of industrially important $\alpha$-amylases. J. Appl. Glycosci. 46: 199-206.   DOI