International Journal of Biological Macromolecules 49 (2011) 103–112 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Comparative analysis of enzymatic stability and amino acid sequences of thermostable alkaline proteases from two haloalkaliphilic bacteria isolated from Coastal region of Gujarat, India Megha K. Purohit, Satya P. Singh ∗ Department of Biosciences, Saurashtra University, Rajkot 360005, India article info Article history: Received 5 March 2011 Received in revised form 1 April 2011 Accepted 4 April 2011 Available online 12 April 2011 Keywords: Haloalkaliphilic bacteria Alkaline proteases Protein purification Thermostability abstract Thermostable alkaline proteases from two haloalkaliphilic bacteria, Oceanobacillus iheyensis O.M.A 1 8 (EU680961) and Haloalkaliphilic bacterium O.M.E 1 2 (EU680960) were studied for enzymatic properties and amino acid sequences in comparative manner. The bacteria were isolated from salt enriched soil located in Okha, Coastal Gujarat, India. The unique aspect of the study was that alkaline protease from Haloalkaliphilic bacterium O.M.A 1 8 optimally catalyzed the reaction over a wide range of temperature, 50–90 ◦ C, with a half-life of 36 h at 90 ◦ C. The molecular weights of O.M.A 1 8 and O.M.E 1 2 were 35 kDa and 25 kDa, respectively. The enzyme secretion was over the broader range of pH 8–11, with an optimum at 11. The alkaline proteases from the two haloalkaliphilic strains isolated from the same site reflected quite different characteristics features. To the best of our knowledge, we have not come across with any such report on the thermal stability of alkaline proteases from haloalkaliphiles. Amino acid sequences for both enzymes were deduced from the nucleotide sequences of their corresponding genes followed by the analysis of physico-chemical properties of the enzymes. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Bacteria secrete variety of enzymes, many of them being com- mercially significant. Beside, the patterns of enzyme secretion and characteristics may also reflect on the population heterogeneity in a particular extreme habitat. Proteases constitute one of the most important groups of industrial enzymes and account for about 60% of the total worldwide enzyme sales [1]. Bacteria display large vari- ations in optimum growth temperatures, often reflected in thermal stabilities of their extracellular enzymes. Over the years, Bacillus species have emerged as prolific producers of extracellular pro- teases with a potential for wide range of applications, in detergent, food, pharmaceutical, leather and chemical industries [2–15]. Thermostable enzymes are of special interest for industrial applications due to their stability under typical operation con- ditions; such as high temperatures and wide pH range. The thermophilic proteases catalyze the reaction and maintain the sta- bility at higher temperatures. In addition, higher temperatures can accelerate the reaction rates, increase the solubility of non- gaseous reactants and products and decrease the incidence of microbial contamination by mesophilic organisms. Many ther- ∗ Corresponding author. Tel.: +91 281 2586419; fax: +91 281 2586419. E-mail addresses: satyapsingh125@gmail.com, satyapsingh@yahoo.com (S.P. Singh). mophiles, such as Bacillus stearothermophilus, Thermus aquaticus, Bacillus licheniformis, Bacillus pumilus and Thermoanaerobacter yon- seiensis, produce a variety of thermostable extracellular proteases [9,16–20]. It has been known that enzymes from thermophilic bac- teria are unusually thermostable, while possessing other properties identical with enzymes found in mesophilic bacteria [21]. Usu- ally, alkaline proteases used in detergents are from thermophiles, having optimum temperatures between 50 and 70 ◦ C [22–24], and stability in the range of 37–70 ◦ C [25]. An alkaline serine protease from Bacillus sp. was highly ther- mostable and retained 100% activity at 60–70 ◦ C for 350–400 min [23]. According to some reports, salt enhanced the thermostability of alkaline proteases. Similarly, Ca 2+ and polyethylene glycol also plays a very important role in enhancing the temperature stability of the enzymes [10–13,26]. The thermostability of enzymes is understood to be a character- istic due to structure of the protein itself [27–31]. The sequencing, structure, and mutagenesis information accumulated during the last 20 years have confirmed that hydrophobicity [28], hydrogen bond, ion pairs and hydrophobic interactions [32], decrease in the uncharged polar residues and increase in charged polar residues in the polypeptide chain of the thermophilic protein contributed sig- nificantly in protein stability at higher temperatures and solvents [33–36]. Now a days, enzyme producing industries use cloning and expression as one of the approaches to obtain high quantity of bio- catalysts [26,37,38]. Protein engineering could be considered as 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.04.001