Process Biochemistry 49 (2014) 61–68 Contents lists available at ScienceDirect Process Biochemistry jo ur nal home p age: www.elsevier.com/locate/procbio Cloning, over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A 1 8 and Haloalkaliphilic bacterium O.M.E 1 2 Megha K. Purohit 1 , 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 28 February 2013 Received in revised form 12 July 2013 Accepted 16 July 2013 Available online 24 July 2013 Keywords: Recombinant enzyme Alkaline protease Haloalkaliphiles Cloning and over-expression 3-D structure Structure and function relationship a b s t r a c t Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A 1 8 and Haloal- kaliphilic bacterium O.M.E 1 2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 ◦ C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Proteases are the largest selling enzymes, accounting for about 60% of the global enzyme market [1–4]. Alkaline proteases are produced by a wide range of organisms; bacteria, moulds, yeasts and mammalian tissues [5]. The microbial alkaline proteases are commercially the most viable enzymes and are derived from the various strains of Bacillus sp. [6–8]. Most of the studies on the haloalkaliphilic bacteria have focused on the diversity and molec- ular phylogeny and only limited reports are available on their enzymatic and other biotechnological potential. Genes from some extremophiles have been cloned and over-expressed in different hosts to obtain large quantities of the recombinant enzymes [9,10]. It is necessary and interesting to compare the folding and func- tioning of the recombinant enzyme to its counterpart produced in native bacteria. Therefore, during the recent years, gene cloning from extremophiles into mesophilic host has gathered consid- erable attention. However, with particular reference to alkaline ∗ Corresponding author. Tel.: +91 281 2586419; fax: +91 281 2586419. E-mail addresses: satyapsingh@yahoo.com, satyapsingh125@gmail.com (S.P. Singh). 1 Current address: Department of Biosciences, Veer Narmad South Gujarat University, Surat, India. proteases from halophiles and haloalkaliphiles, only few enzymes have been purified and characterized [9]. The saline habitats along the Gujarat Coast in Western India exhibits significant diversity of natural microbial flora. However, it is rarely explored and investi- gated for molecular biological properties and enzymatic potential [2–4,10–14]. Developments in molecular approaches to improve the cloning and expression of genes, from halophilic and other extremophilic organisms, for enhanced solubilization of the expressed proteins would add to the prospect of enzyme-driven catalysis [13–19]. In this report, we describe the cloning and expression of two alkaline protease genes from haloalkaliphilic bacteria into Escherichia coli as host, followed by the characterization of the recombinant enzymes to establish the structure and function relationship. 2. Materials and methods 2.1. Bacterial strains and plasmids for cloning and expression study The bacterial strains used for cloning and expression were E. coli TOP10 and BL21 (DE3) (Invitrogen, USA), while pET21a+ (Invitrogen, USA) plasmid was used as vector. 2.2. Sample collection The haloalkaliphilic bacteria were isolated from salt enriched soil near a salt pan located in Okha-Madhi (Latitude 22.20 N, Longitude 70.05 E), the coastal region 1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2013.07.009