Enzyme stability, thermodynamics and secondary structures of α-amylase as probed by the CD spectroscopy

An amylase of a thermophilic bacterium, Bacillus sp. TSSC-3 (GenBank Number, EU710557) isolated from the Tulsi Shyam hot spring reservoir (Gujarat, India) was purified to the homogeneity in a single step on phenyl sepharose 6FF. The molecular weight of the enzyme was 25 kD, while the temperature and pH optima for the enzyme catalysis were 80 degrees C and 7, respectively. The purified enzyme was highly thermostable with broad pH stability and displayed remarkable resistance against surfactants, chelators, urea, guanidine HCl and various solvents as well. The stability and changes in the secondary structure of the enzyme under various extreme conditions were determined by the circular dichroism (CD) spectroscopy. The stability trends and the changes in the alpha-helices and beta-sheets were analyzed by Mean Residual Ellipticity (MRE) and K2D3. The CD data confirmed the structural stability of the enzyme under various harsh conditions, yet it indicated reduced alpha-helix content and increased beta-sheets upon denaturation. The thermodynamic parameters; deactivation rate constant, half-life, changes in entropy, enthalpy, activation energy and Gibbs free energy indicated that the enzyme substrate reactions were highly stable. The overall profile of the enzyme: high thermostability, alkalitolerance, calcium independent nature, dextrose equivalent values and resistance against chemical denaturants, solvents and surfactants suggest its commercial applications. (C) 2015 Elsevier B.V. All rights reserved.