Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 889, Issue -, Pages 61-68Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2012.01.031
Keywords
Salt-tolerant alkaliphilic actinomycetes; Thermostability; Alkaline protease; Enzyme purification; Enzyme kinetics; Thermodynamics; Denaturation constant
Funding
- UGC, New Delhi, India
- Saurahtra University, Rajkot, India
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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.
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