Differential scanning calorimetric studies of a Bacillus halodurans α-amylase

The thermal unfolding of Amy 34, a recombinant α-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (Tm) of 70.8 °C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The Tm increased by 5 °C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (ΔHcal), however, at higher concentrations of CaCl2 (up to 100-fold), ΔHcal was found to decrease, accompanied by a decrease in entropy change (ΔS), while the Tm steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in Tm by 10.4 °C. Tm was also decreased to 61.1 °C and 65.9 °C at pH 6.0 and pH 11.0, respectively.