title: Role of Val289 residue in the alpha-amylase of Bacillus amyloliquefaciens MTCC 610: an analysis by site directed mutagenesis.
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.
authors.affiliation: Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai-625 021, India.
authors.forename: R
authors.initials: R
authors.lastname: Priyadharshini
authors.forename: D
authors.initials: D
authors.lastname: Hemalatha
authors.forename: P
authors.initials: P
authors.lastname: Gunasekaran
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D019295:Computational Biology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014633:Valine / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000516:alpha-Amylases; D014633:Valine