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 °C in 4 M NaCl and 30% Na-glutamate, with significant stability at 60–80 °C in Na-glutamate. Deactivation rate constant (Kd) increased and half life (t1/2) decreased with the increasing temperatures from 37 to 80 °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 °C in 30% Na-glutamate with Kd 4.11 and t1/2 168.64 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, H2O2, β-mercaptoethanol and different surfactants enhanced the activity.