Engineering of a Bacillus alpha-amylase with improved thermostability and calcium independency.
Successful industrial use of amylases requires that they are sufficiently stable and active at application conditions, e.g., at high temperature in starch-liquefaction process. In the present study, site-directed mutagenesis was used to enhance the thermal stability and calcium independency of a mesophilic alpha-amylase from Bacillus megaterium WHO. Mutations (A53S and H58I) were designed at the calcium-binding site based on the sequence alignment. Kinetic and thermostability parameters of the mutants were analyzed and compared with that of the wild type. In the presence of calcium, the affinity of the enzymes (wild type and mutants) toward starch was increased. In comparison to the wild type, calcium ion had more effect on the catalytic efficiency, k (cat)/K (m), and half-life (at 60 degrees C) of A53S mutant. In A53S, the dependence of half-life on calcium concentration showed that the enhanced calcium binding is likely to be responsible for the increased stability. In contrast, calcium-independent mutant (H58I) possessed high thermostability. In addition, thermodynamic parameters of amylolytic reaction exhibited an increase in the activation energy and the entropy of the system. Kinetics of irreversible thermal inactivation suggests that the activation energy increased by 1.4-fold in the most stable variant.
Ghollasi
Marzieh
M
Department of Biochemistry and Biophysics, Faculty of Biological Science, Tarbiat Modares University, PO Box: 14115-175, Tehran, Iran.
Khajeh
Khosro
K
Naderi-Manesh
Hossein
H
Ghasemi
Atiyeh
A
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D019277:Entropy; D004795:Enzyme Stability / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D056811:Unfolded Protein Response; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D000516:alpha-Amylases; D002118:Calcium