Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilus alpha-amylase.

Bacillus stearothermophilus NCIB 11412 produces a highly thermostable alpha-amylase. The enzyme displayed half-lives of irreversible thermoinactivation at 90 degrees C of 1.9 min and 12.5 min at pH 5.0 and pH 8.0, respectively. Molecular mechanisms of irreversible thermoinactivation were investigated. At both pH 5.0 and pH 8.0 irreversible thermoinactivation was due to heat-induced breakdown of non-covalent interaction within the protein molecule, resulting in unfolding and consequent formation of altered structures. Hydrophobic interactions were shown to be the most important non-covalent mechanisms involved in this phenomenon. Although not dramatically effecting the rates of irreversible thermoinactivation, electrostatic interactions, including hydrogen bonding, were also shown to have a contributory role in this process. At pH 8.0 a covalent mechanism, that of oxidation of thiols was also shown to be of secondary importance to hydrophobic interactions in the irreversible thermoinactivation of this enzyme.