Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Role of Val289 Residue in the $\alpha$-Amylase of Bacillus amyloliquefaciens MTCC 610: An Analysis by Site Directed Mutagenesis

  • Priyadharshini, R. (Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University) ;
  • Hemalatha, D. (Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University) ;
  • Gunasekaran, P. (Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University)
  • Received : 2009.10.01
  • Accepted : 2009.11.03
  • Published : 2010.03.31
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Abstract

The Val289 residue in the $\alpha$-amylase of Bacillus amyloliquefaciens, which is equivalent to the Ala289 and Val286 residues in the $\alpha$-amylases of B. stearothermophilus and B. licheniformis, respectively, was studied by site-directed mutagenesis. This residue was substituted with 10 different amino acids by random substitution of the Val codon. In these mutant $\alpha$-amylases, Val289 was substituted with Ile, Tyr, Phe, Leu, Gly, Pro, Ser, Arg, Glu, and Asp. Compared with the wild-type $\alpha$-amylase, the mutant $\alpha$-amylase Val289Ile showed 20% more hydrolytic activity, whereas Val289Phe and Val289Leu showed 50% lesser activity. On the other hand, the mutant $\alpha$-amylases Val289Gly, Val289Tyr, Val289Ser, and Val289Pro showed less than 15% activity. The substitution of Val289 with Arg, Asp, or Glu resulted in complete loss of the $\alpha$-amylase activity. Interestingly, the mutant $\alpha$-amylase Val289Tyr had acquired a transglycosylation activity, which resulted in the change of product profile of the reaction, giving a longer oligosaccharide.

Keywords

References

  1. Arnold, K., L. Bordoli, J. Kopp, and T. Schwede. 2006. The SWISS-MODEL Workspace: A Web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201. https://doi.org/10.1093/bioinformatics/bti770
  2. DeLano, W. L. 2003. PyMOL Reference Manual. DeLano Scientific LLC, San Carlos, CA.
  3. Guex, N. and M. C. Peitsch. 1997. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714-2723. https://doi.org/10.1002/elps.1150181505
  4. Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280: 309-316.
  5. Igarashi, K., Y. Hatada, H. Hagihara, K. Saeki, M. Takaiwa, T. Uemura, et al. 1998. Enzymatic properties of a novel liquefying $\alpha$-amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences. Appl. Environ. Microbiol. 64: 3282-3289.
  6. Janecek, S. 2002. How many conserved sequence regions are there in the $\alpha$-amylase family? Biologia 57 (Suppl. 11): 29-41.
  7. Kuriki, T., H. Kaneko, M. Yanase, H. Takata, J. Shimada, S. Handa, T. Takada, H. Umeyama, and S. Okada. 1996. Controlling substrate preference and transglycosylation activity of neopullulanase by manipulating steric and hydrophobicity in active center. J. Biol. Chem. 271: 17321-17329. https://doi.org/10.1074/jbc.271.29.17321
  8. MacGregor, E. A. 2005. An overview of clan GH-H and distantly-related families. Biologia 60 (Suppl. 16): 5-12.
  9. Miller, L. H. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 246-248.
  10. Nakajima, R., T. Imanaka, and S. Aiba. 1985. Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene. J. Bacteriol. 163: 401-406.
  11. Pandey, A., P. Nigam, C. R. Soccol, V. T. Soccol, D. Singh, and R. Mohan. 2000. Advances in microbial amylases. Biotechnol. Appl. Biochem. 31: 135-152. https://doi.org/10.1042/BA19990073
  12. Rivera, M. H., M. A. Lopez, X. Soberon, and G. Saab-Rincon. 2003. $\alpha$-Amylase from Bacillus licheniformis mutants near to the catalytic site: Effects on hydrolytic and transglycosylation activity. Protein Eng. 16: 505-514. https://doi.org/10.1093/protein/gzg060
  13. Saab-Rincon, G., R. G. Del, R. I. Santamaria, M. A. Lopez, and X. Soberon. 1999. Introducing transglycosylation activity in a liquefying $\alpha$-amylase. FEBS Lett. 453: 100-106. https://doi.org/10.1016/S0014-5793(99)00671-7
  14. Schwede, T., J. Kopp, N. Guex, and M. C. Peitsch. 2003. SWISS-MODEL - An automated protein homology-modeling server. Nucleic Acids Res. 31: 3381-3385. https://doi.org/10.1093/nar/gkg520
  15. Svensson, B., M. T. Jensen, H. Mori, K. S. Bak Jensen, I. Bnsager, et al. 2002. Fascinating facets of function and structure of amylolytic enzymes of glycoside hydrolase family 13. Biologia 57 (Suppl. 11): 5-19.
  16. Takata, H., T. Kuriki, S. Okada, Y. Takesada, M. Iizuka, N. Minamiura, and T. Imanaka. 1992. Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1,4) and alpha-(1,6)-glucosidic linkages. J. Biol. Chem. 267: 18447-18452.
  17. Takkinen, K., R. F. Pettersson, N. Kalkkinen, I. Palva, H. Soederlund, and L. Kaeaeriaeinen. 1983. Amino acid sequence of alpha-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene. J. Biol. Chem. 258: 1007-1013.
  18. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24: 4876-4882.
  19. Yuuki, T., T. Nomura, H. Tezuka, A. Tsuboi, H. Yamagata, N. Tsukagoshi, and S. Udaka. 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: 1147-1156.