title: Thermodynamics of a Ca(2+)-dependent highly thermostable alkaline protease from a haloalkliphilic actinomycete.
abstract: An alkaline protease from salt-tolerant alkaliphilic actinomycetes, Nocardiopsis alba OK-5 was purified by a single-step hydrophobic interaction chromatography and characterized. The purified protease with an estimated molecular mass of 20 kDa was optimally active at 70 °C in 0-3 M NaCl and 0-100 mM Ca(2+) displaying significant stability at 50-80 °C. The enzyme was stable at 80 °C in 100 mM Ca(2+) with Kd of 17 × 10(-3) and t1/2 of 32 min. The activation energy (Ea), enthalpy (ΔH*), and entropy (ΔS*) for the protease deactivation calculated in the presence of 200 mM Ca(2+) were 38.15 kJ/mol, 35.49 kJ/mol and 183.48 J/mol, respectively. The change in free energy (ΔG*) for protease deactivation at 60 °C in 200 mM Ca(2+) was 95.88 kJ/mol. Decrease in ΔH* reflected reduced cooperativity of deactivation and unfolding. The enzyme was intrinsically stable that counteracted heat denaturation by a weak cooperativity during the unfolding. Further, the enzyme was highly stable in the presence of various cations, surfactants, H2O2, β-mercaptoethanol, and commercial detergents. The compatibility of the enzyme with various cations, surfactants, and detergent matrices suggests its suitability as an additive in the detergents and peptide synthesis.
authors.affiliation: UGC CAS Department of Biosciences, Saurashtra University, Rajkot 360005, India.
authors.forename: S D
authors.initials: SD
authors.lastname: Gohel
authors.affiliation: UGC CAS Department of Biosciences, Saurashtra University, Rajkot 360005, India. Electronic address: satyapsingh@yahoo.com.
authors.forename: S P
authors.initials: SP
authors.lastname: Singh
mesh_terms: D039903:Actinobacteria / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D055162:Biocatalysis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability / Q000187:drug effects; D005658:Fungi / Q000737:chemistry / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D012965:Sodium Chloride / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D013501:Surface-Active Agents; D012965:Sodium Chloride; D006861:Hydrogen Peroxide; D010450:Endopeptidases; C087822:alkaline protease; D002118:Calcium
keywords: Biocatalysis; Detergent stable thermophilic enzyme.; Enzyme deactivation; Filamentous bacteria; Protease; Thermodynamics