title: Purification strategies, characteristics and thermodynamic analysis of a highly thermostable alkaline protease from a salt-tolerant alkaliphilic actinomycete, Nocardiopsis alba OK-5.
abstract: An alkaline protease from salt tolerant alkaliphilic actinomycetes, Nocardiopsis alba strain OK-5 was purified to homogeneity by 27 and 13 fold with a yield of 35 and 13% using two-steps and one-step method, respectively. The purification methods involved hydrophobic interaction on phenyl sapharose matrix. The apparent molecular mass was 20 kDa. The temperature optimum shifted from 70 to 80°C in 4M NaCl and 30% Na-glutamate, with significant stability at 60-80°C in Na-glutamate. Deactivation rate constant (K(d)) increased and half life (t(1/2)) decreased with the increasing temperatures from 37 to 80°C. The order of stability was: 30% Na-glutamate>4M NaCl>2M NaCl>0M NaCl. The enzyme was stable even at 80°C in 30% Na-glutamate with K(d) 4.11 and t(1/2) 168.64 min. The activation energies (E), enthalpy (ΔH*) and entropy (ΔS*) for protease deactivation in with Na-glutamate were 31.97 kJ/mole, 29.23 kJ/mole and -211.83 J/mole, respectively. The change in free energy (ΔG*) for protease deactivation at 60°C in 30% Na-glutamate was 101.70 kJ/mole. Protease had the highest activity and stability at pH 10-11. While the enzyme was highly resistant against chemical denaturation, it had varied responses to metal ions. Complete inhibition by PMSF confirmed serine nature of the protease. Na-glutamate, H(2)O(2), β-mercaptoethanol and different surfactants enhanced the activity.
authors.affiliation: Department of Biosciences, Saurashtra University, Rajkot, India.
authors.forename: Sangeeta D
authors.initials: SD
authors.lastname: Gohel
authors.forename: Satya P
authors.initials: SP
authors.lastname: Singh
mesh_terms: D039903:Actinobacteria / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000379:methods; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D010802:Phylogeny; D055049:Salt Tolerance / Q000502:physiology; D012965:Sodium Chloride / Q000494:pharmacology; D012970:Sodium Glutamate / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D013501:Surface-Active Agents; D012965:Sodium Chloride; D010450:Endopeptidases; C087822:alkaline protease; D012970:Sodium Glutamate