Improved thermostability of a Bacillus alpha-amylase by deletion of an arginine-glycine residue is caused by enhanced calcium binding.
alpha-Amylase from alkaliphilic Bacillus KSM-1378 (LAMY) is a novel semi-alkaline enzyme which has a high specific activity, a value 5-fold higher than that of a Bacillus licheniformis enzyme at alkaline pH. Thermostability of this enzyme could be improved by deletion of the Arg181-Gly182 residue by means of site-directed mutagenesis. The wild-type and engineered LAMYs were very similar with respect to specific activity, pH-activity curve, temperature-activity curve, susceptibility to inhibitors, and pattern of hydrolysis products from soluble starch and maltooligosaccharides. However, the engineered enzyme also acquired increased pH stability and resistance to sodium dodecyl sulfate and especially chelating reagents, such as ethylenediaminetetraacetate and ethyleneglycol-bis (beta-aminoethylether)tetraacetate. This is the first report that thermostability of alpha-amylase is improved by enhanced calcium binding to the enzyme molecule.
Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi, 321-3497, Japan.
K
K
Igarashi
Y
Y
Hatada
K
K
Ikawa
H
H
Araki
T
T
Ozawa
T
T
Kobayashi
K
K
Ozaki
S
S
Ito
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017421:Sequence Analysis; D017384:Sequence Deletion / Q000235:genetics; D013213:Starch / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry*
D002614:Chelating Agents; D003902:Detergents; D011994:Recombinant Proteins; D013213:Starch; D000516:alpha-Amylases; D002118:Calcium