Journal of Chromatography B, 889–890 (2012) 61–68 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l ho me page: www.elsevier.com/locate/chromb Purification strategies, characteristics and thermodynamic analysis of a highly thermostable alkaline protease from a salt-tolerant alkaliphilic actinomycete, Nocardiopsis alba OK-5 Sangeeta D. Gohel, Satya P. Singh ∗ Department of Biosciences, Saurashtra University, Rajkot 360005, India a r t i c l e i n f o Article history: Received 15 June 2011 Accepted 26 January 2012 Available online 3 February 2012 Keywords: Salt-tolerant alkaliphilic actinomycetes Thermostability Alkaline protease Enzyme purification Enzyme kinetics Thermodynamics Denaturation constant a b s t r a c t 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 (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 > 4 M NaCl > 2 M NaCl > 0 M 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 (H*) and entropy (S*) 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 (G*) 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 , -mercaptoethanol and different surfactants enhanced the activity. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Studies on extremophiles led to the search for many enzymes that exhibited much needed features. Besides their biotechnolog- ical prospects, the enzymes from extremophiles may also provide unique models to understand the biochemical and molecular basis of the adaptation under extreme conditions. Though enzymes from halophilic archaea and bacteria are fairly well characterized recently [1–4], similar attention has not been focused on haloal- kaliphilic actinomycetes [5–9]. While, antibiotics have been the major bioactive compounds from actinomycetes, their ability to produce a variety of enzymes has been explored only in limited sense. The enzymatic spectrum of such organisms, though not sys- tematically explored, appears quite promising as indicated by some preliminary indications. We explored haloalkaliphilic proteases from actinomycetes as these enzymes occupy a pivotal position with respect to their applications and cellular significance. Property of halophilic proteases severely restricts the choice of purification methods, as they require higher salt for their activity ∗ Corresponding author at: Department of Biosciences, Saurashtra University, Rajkot 360005, Gujarat, India. Tel.: +91 281 2586419; fax: +91 281 2586419. E-mail address: satyapsingh@yahoo.com (S.P. Singh). and stability, making most of the conventional procedures unsuit- able. The methods used for purification of haloarchaeal proteases include concentration of the enzyme by ethanol precipitation or ultra filtration followed by affinity and gel filtration chromatog- raphy. However, many steps make the method cumbersome and adversely affect the yield of the purified enzyme. Therefore, one step purification of the concentrated enzyme by hydrophobic inter- action provides a method of choice [10–12]. The activity and stability of enzymes are important parameters to determine the economic feasibility in industrial processes. High stability is generally considered an economic advantage because of reduced enzyme turnover [13]. Before proceeding to develop suit- able protease enzyme formulations, accumulating information on the stability of enzymes in different conditions is necessary. Studies on the thermodynamic stability of enzymes have provided funda- mental insights into the factors that determine enzyme stability [13,14]. However, for actinomycetes, thermodynamic properties of the purified protease have not been described in the literature. Thus, the present investigation considered different thermody- namic approaches; deactivation kinetics, H*, S*, E and G* to understand the behavior of protease at different temperatures and salts. Halophilic eubacteria accumulate organic compatible solutes such as sucrose, mannitol, trehalose, glycerol, betaine, proline 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2012.01.031