title: Enzyme stability, thermodynamics and secondary structures of α-amylase as probed by the CD spectroscopy.
abstract: 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 25kD, while the temperature and pH optima for the enzyme catalysis were 80°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 α-helices and β-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 α-helix content and increased β-sheets upon denaturation. The thermodynamic parameters; deactivation rate constant, half-life, changes in entropy, enthalpy, activation energy and Gibb's 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.
authors: Department of Biosciences, Saurashtra University, Rajkot 360005, Gujarat, India.
authors: B A
authors: BA
authors: Kikani
authors: Department of Biosciences, Saurashtra University, Rajkot 360005, Gujarat, India. Electronic address: satyapsingh@yahoo.com.
authors: S P
authors: SP
authors: Singh
mesh_terms: D001407:Bacillus / Q000201:enzymology; D002412:Cations / Q000737:chemistry; D002614:Chelating Agents / Q000494:pharmacology; D002845:Chromatography; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017433:Protein Structure, Secondary; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D002412:Cations; D002614:Chelating Agents; D013501:Surface-Active Agents; D000516:alpha-Amylases
keywords: CD spectroscopy; Thermodynamics; Thermostability