Thermal stability of alpha-amylase in aqueous cosolvent systems

The activity and thermal stability of a-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 +/- 2 degrees C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (T-m)(app) and activation energy (E-a) of alpha-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the,presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (T-m)(app) were 20 degrees C, 14 degrees C, 13 degrees C and 9 degrees C, respectively. The E-a of thermal denaturation of alpha-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.