title: Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase.
abstract: Asparagine deamidation is one of the important determinants of protein thermostability. Here, structure based mutagenesis has been done in order to probe the role of Asn residues in thermostability of a Ca independent Bacillus sp. KR-8104 α-amylase (BKA). Residues involved in potential deamidation processes have been selected and replaced using a site directed mutagenesis. Fourteen different variants were tested for thermostability by measuring residual activities after incubation at high temperature. In comparison to the wild-type enzyme, four mutated variants are able to increase the half life of the protein at high temperatures. The highest stabilization resulted from the substitution of asparatate in place of asparagine at position 112, leading to a nearly fivefold increase of the enzyme's half-life at 70°C. Also replacement of Asn129 to aspartic acid and Asn312 to serine markedly increased the half-life of the enzyme at 70°C indicating that the deamination of these residues may have a deleterious effect on BKA.
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Mahsa
authors: M
authors: Rahimzadeh
authors: Khosro
authors: K
authors: Khajeh
authors: Manoochehr
authors: M
authors: Mirshahi
authors: Mahmood
authors: M
authors: Khayatian
authors: Robert
authors: R
authors: Schwarzenbacher
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine; D001407:Bacillus / Q000201:enzymology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D001216:Asparagine; D000516:alpha-Amylases