Purification strategies, characteristics and thermodynamic analysis of a highly thermostable alkaline protease from a salt-tolerant alkaliphilic actinomycete, Nocardiopsis alba OK-5

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 degrees C in 4 M NaCl and 30% Na-glutamate, with significant stability at 60-80 degrees 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 degrees C. The order of stability was: 30% Na-glutamate > 4 M NaCl > 2 M NaCl > 0 M NaCl. The enzyme was stable even at 80 degrees C in 30% Na-glutamate with K-d 4.11 and t(1/2) 168.64 min. The activation energies (E), enthalpy (Delta H*) and entropy (Delta S*) for protease deactivation in with Na-glutamate were 31.97 kJ/mole, 29.23 kJ/mole and -211.83J/mole, respectively. The change in free energy (Delta G*) for protease deactivation at 60 degrees 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, H2O2, beta-mercaptoethanol and different surfactants enhanced the activity. (C) 2012 Elsevier B.V. All rights reserved.