A differential behavior of α-amylase, in terms of catalytic activity and thermal stability, in response to higher concentration CaCl2

A differential relationship was observed between thermal stability and catalytic activity of α-amylase in the presence of different concentrations of CaCl2. The enzyme displays optimum catalytic activity in the presence of 1.0-2.0 mM CaCl2. Further addition of CaCl2 leads to inhibition of the enzyme, however, at the same time the enzyme gains an additional resistance against thermal denaturation. It was evident that the enzyme is thermodynamically more stable (compared to the active enzyme) in the presence of inhibitory concentration of CaCl2. For example, the thermal transition temperature (Tm) of optimally active α-amylase was found to be 64 ± 1 °C, whereas, for the less active enzyme (in the presence 10 mM CaCl2) the value was determined to be 71 ± 1 °C. Similarly, the activation energy of thermal inactivation (Ea) was found to be 228 ± 12 kJ/mol and 291 ± 15 kJ/mol for the optimally active enzyme and the enzyme in the presence of 10 mM CaCl2, respectively. Biophysical analysis of different states of the enzymes in response to variable calcium level indicates no significant change in the secondary structure in response to different concentration of CaCl2, however the less active but thermodynamically stable enzyme (in the presence of higher concentration of CaCl2) was shown to have relatively more compact structure. The results suggest that the enzyme has separate catalytic and structure stabilizing domains and they significantly vary in their functional attributes in response to calcium level.