title: Improvement of thermal stability of a mutagenised α-amylase by manipulation of the calcium-binding site.
abstract: Site-directed mutagenesis of an α-amylase isolated from Bacillus megaterium WHO has been performed to evaluate the roles of the calcium binding site residues in enzyme thermostability. The strategy used was to replace residues in the hypothetical calcium binding loops of B. megaterium WHO α-amylase (BMW-amylase) by equivalent positions at Halothermothrix orenii α-amylase (AmyA) as a thermophilic amylase by QuikChange site directed mutagenesis. Asn-75, Ser-76, and His-77 were mutated in the second calcium binding site which resulted in an increase in thermostability. All mutants retained their hydrolytic activity although their kcat parameter decreased in compare to the wild type and in the presence of calcium ions. In S76P and H77E, the Km for starch decreases while overall activity (kcat/Km) was increased. In the presence of calcium, conversion of His-77 to Glu resulted in a 4-fold enhancement in enzyme half life and a 9°C upward shift in T50, which was observed in compare to the wild type. Further analysis suggested the H77E mutant as the most stable which increased the affinity of the enzyme for calcium ion and its optimum temperature was 5°C higher than the wild type.
authors: Department of Cell and Molecular biology, Faculty of Biological Science, Kharazmi University, P.O. Box: 31979-37551, Tehran, Iran.
authors: Marzieh
authors: M
authors: Ghollasi
authors: Maryam
authors: M
authors: Ghanbari-Safari
authors: Khosro
authors: K
authors: Khajeh
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D006207:Half-Life; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D000516:alpha-Amylases; D002118:Calcium
keywords: Bacillus megaterium; Calcium; Mutagenesis; Thermostability; α-Amylase