Stabilization of α-Amylase, the Key Enzyme in Carbohydrates Properties Alterations, at Low pH

Abstract

The present study correlates the mechanism of α-amylase denaturation under acidic condition and its structural stabilization in presence of selected cosolvents (sorbitol, sucrose, trehalose, and glycerol). The objective of the present study was to minimize the enzyme inactivation at lower pH by stabilizing the enzyme structure. The above cosolvents were found to be an effective stabilizer of α-amylase against denaturation at extreme low pH. The optimum activity of α-amylase was found to be in the pH range of 4.5 to 7. Decreasing the pH of enzyme solution below this range results in a decrease in enzyme activity. The intrinsic and ANS fluorescence indicated the gradual unfolding of protein below pH 4 and resulted in exposure of hydrophobic cluster. The pH-induced unfolding also resulted in protein aggregation. The intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence, acrylamide quenching, near and far UV-CD spectra of α-amylase at lower pH (pH 2.25) indicated the complete loss of tertiary structure and substantial loss of secondary structure. Cosolvents were shown to prevent the acid induced unfolding of the enzyme to a certain extent and help in retaining the secondary structure of enzyme equivalent to native state. Among all the cosolvents used, sorbitol was proven to be the most efficient stabilizer. At lower pH, in presence of cosolvents the enzyme was shown to retain significant amount of secondary structure and poorly defined tertiary structure. This structure resembles the molten globule of the protein, which has substantial amount of secondary structure but poorly defined tertiary structure.