International Journal of Biological Macromolecules 56 (2013) 20–27 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules jo u rn al hom epa ge: www.elsevier.com/locate/ijbiomac Characteristics and thermodynamics of a thermostable protease from a salt-tolerant alkaliphilic actinomycete S.D. Gohel, S.P. Singh ∗ Department of Biosciences, Saurashtra University, Rajkot 360 005, Gujarat, India a r t i c l e i n f o Article history: Received 8 November 2012 Received in revised form 23 January 2013 Accepted 24 January 2013 Available online 5 February 2013 Keywords: Salt-tolerant alkaliphilic actinomycetes Thermostable alkaline protease Single step purification Enzyme kinetics Thermodynamics a b s t r a c t An alkaline serine protease from a newly isolated salt-tolerant alkaliphilic actinomycetes, Brachys- treptospora xinjiangensis OM-6 was purified with 35- and 26-fold purification and 47% and 22% yield employing two steps and one step methods, respectively. The enzyme was quite stable at 80 ◦ C in 30% Na-glutamate with the deactivation rate constant (K d ) 8.66 and half life (t 1/2 ) 80.04 min. The activation energies (E), enthalpy (H*), entropy (S*) and change in free energy (G*) for the protease deactiva- tion were calculated in the presence of 30% Na-glutamate and correlated with the enzyme stability. The thermodynamic analysis corresponded the trends of the enzyme stability and inactivation. The enzyme retained high activity and significant stability at higher salt, temperature, range of pH and metal ions. The enzyme was extremely resistant against urea denaturation, oxidizing and reducing agents and surfactants, a finding which is rather unique and restricted to only few proteins. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Proteases are established in enzyme catalysis and have been the subject of numerous texts and reviews. The majorities of halophilic proteases, however, have been studied from halophilic archaea and bacteria [1–5], while representation from haloalka- liphilic actinomycetes in this context is quite restricted [6–11]. Despite their advantages, the application of halophilic proteases is further constrained because of their limited stability under extremes of temperature, pH and ionic strength. The available array is still not sufficient to meet the ever increasing demand for suitable proteases. Halophilic proteases have several applications in food, leather, detergent and antifouling coating industries as well as pep- tide synthesis in organic media. Haloarchaeal proteases catalyze the reaction at 4–5 M NaCl, losing activity rapidly when exposed to low salt concentrations [12]. This property of enzyme severely restricts the choice of purification methods, making the majority of the conventional purification procedures unsuitable. The methods used for purification of haloarchaeal proteases include concentrat- ing the enzyme by ethanol precipitation or ultra filtration followed by affinity chromatography and gel filtration. However, many steps make the method more cumbersome and hence, single step purifi- cation by hydrophobic interaction chromatography is a better choice [13–15]. ∗ Corresponding author. Tel.: +91 281 2586419; fax: +91 281 2586419. E-mail address: satyapsingh@yahoo.com (S.P. Singh). Most of the halophilic proteases belong to serine protease fam- ily, dependent on high salt concentrations for structural stability and display optimum activity at high salt, neutral to basic pH and temperatures 37–50 ◦ C. However, literatures describing halophilic proteases with alkaliphilic and thermophilic properties are quite limited, especially from haloalkaliphilic actinomycetes [16]. The activity and stability of enzymes are important parameters which codetermine the economic feasibility of applying protease in indus- trial processes. High stability is generally considered an economic advantage because of reduced enzyme turnover [17]. A number of strategies are applied to increase the stability of protease enzymes such as mutation, chemical modification at active site, introduc- tion of disulfide bridges, the optimization of helices and helix caps, immobilization, entropic stabilization, changing pH condi- tion and using various salts. Studies on the thermodynamic stability of enzymes have provided insight into the factors that determine enzyme stability [17]. However, thermodynamic properties of puri- fied proteases, particularly from actinomycetes, are not described. Thus, the present investigation addresses upon the thermodynamic approaches (deactivation kinetics, H*, S*, E and G*) to under- stand the behavior of these enzymes at different temperatures and salts [18]. Halophilic eubacteria accumulate organic compatible solutes such as sucrose, mannitol, trehalose, glycerol, betaine, proline, glu- tamate, etc. to maintain the protein structure. Vidyasagar et al. [19] studied an extracellular protease from Halogeometricum brorin- quense strain TSS101 in the presence of compatible solutes; sucrose, mannitol, glycerol and betaine in absence of NaCl. However, no fur- ther information is available on the stability of this enzyme in these 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.01.028