J. Microbiol. Biotechnol. (2011), 21(2), 129–135 doi: 10.4014/jmb.1007.07064 First published online 22 November 2010 Gene Identification and Molecular Characterization of Solvent Stable Protease from A Moderately Haloalkaliphilic Bacterium, Geomicrobium sp. EMB2 Karan, Ram 1 , Raj Kumar Mohan Singh 2 , Sanjay Kapoor 2 , and S. K. Khare 1 * Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110 021, India Received: July 30, 2010 / Revised: October 21, 2010 / Accepted: November 11, 2010 Cloning and characterization of the gene encoding a solvent-tolerant protease from the haloalkaliphilic bacterium Geomicrobium sp. EMB2 are described. Primers designed based on the N-terminal amino acid sequence of the purified EMB2 protease helped in the amplification of a 1,505-bp open reading frame that had a coding potential of a 42.7-kDa polypeptide. The deduced EMB2 protein contained a 35.4-kDa mature protein of 311 residues, with a high proportion of acidic amino acid residues. Phylogenetic analysis placed the EMB2 gene close to a known serine protease from Bacillus clausii KSM-K16. Primary sequence analysis indicated a hydrophobic inclination of the protein; and the 3D structure modeling elucidated a relatively higher percentage of small (glycine, alanine, and valine) and borderline (serine and threonine) hydrophobic residues on its surface. The structure analysis also highlighted enrichment of acidic residues at the cost of basic residues. The study indicated that solvent and salt stabilities in Geomicrobium sp. protease may be accorded to different structural features; that is, the presence of a number of small hydrophobic amino acid residues on the surface and a higher content of acidic amino acid residues, respectively. Keywords: Halophiles, protease gene, Bacillus clausii, Geomicrobium sp., solvent-tolerant protease Proteases are the earliest-known enzymes and extensively characterized from a variety of sources. The ability to withstand detergents and stability in solvent medium are new attributes pointed out in some of these enzymes [4, 16, 24]. The stability in salt/solvents and the structural features that are responsible for these enzymatic properties are yet to be fully understood. A few proteases from moderately halophilic bacteria have been purified and studied; namely, those from Bacillus sp. no. 21-1 [13], haloalkaliphilic Bacillus sp. Vel [7], Filobacillus sp. RF2-5 [8], Halobacillus sp. SR5-3 [22], Salinivibrio sp. strain AF-2004 [16], haloalkaliphilic bacterium sp. AH-6 [4], and Halobacillus karajensis [15]. Reports on the protease gene are still less documented; for example, halolysin 172P1 from Natrialba asiatica [12], halolysin R4 from Haloferax mediterranei [11], Spt A from Natrinema sp. J7 [30], SVP2 from Salinivibrio sp. strain AF-2004 [17], Nep from Natrialba magadii [3], CPI from Pseudoalteromonas ruthenica [28], and PCP-03 from Pseudoalteromonas sp. SM9913 [32]. Only limited knowledge is available on their three-dimensional structure [20]. Characterization of the biochemical properties in combination with the gene information would be helpful to improve the understanding of halophilic proteases. We have previously reported the isolation of a moderately haloalkaliphilic Geomicrobium sp. EMB2 strain from Sambhar Salt Lake, India. This strain was polyextremic, and thus able to grow in the presence of high salt concentrations as well as alkaline conditions. Furthermore, it secreted a novel protease, which was catalytically active and stable at high concentrations of a wide range of organic solvents. Geomicrobium sp. EMB2 protease was purified to homogeneity by hydrophobic interaction chromatography and found to be a 38-kDa serine protease [14]. While comparing the properties of Geomicrobium sp. EMB2 protease with other known haloalkaliphilic proteases, it became apparent that EMB2 protease differs from other halophiles with respect to higher pH optima, high pH stability, stabilities in surfactant and detergent, and tolerance to organic solvents. The basis of these differences and unique attributes needs to be elucidated. Understanding of such structural principles will provide necessary tools for protein designing. The present study was undertaken to (i) characterize its gene by cloning and sequencing, (ii) carry out an in silico *Corresponding author Phone: +91 11 2659 6533; Fax: +91 11 2658 1102; E-mail: skhare@rocketmail.com, skkhare@chemistry.iitd.ac.in