Production of extracellular halo-alkaline protease from a newly isolated Haloalkaliphilic Bacillus sp. isolated from seawater in Western India R.K. Patel 1 , M.S. Dodia 2 , R.H. Joshi 2 and S.P. Singh 2, * 1 Department of Life Sciences, University of North Gujarat, Patan, India 2 Department of Biosciences, Saurashtra University, 360 005, Rajkot, Gujarat, India *Author for correspondence: Tel.: + 91 281 2586419, Fax: + 91 281 2586419, E-mail: satyapsingh@yahoo.com Received 19 March 2005; accepted 17 August 2005 Keywords: Alkaline proteases, Extremophiles, Haloalkaliphiles, Protease production Summary Haloalkaliphilic, gram positive, aerobic, coccoid Bacillus sp. Po2 was isolated from a seawater sample in Gujarat, India. On the basis of 16s rRNA gene homology, Po2 was 95% related to Bacillus pseudofirmus. A substantial level of extracellular alkaline protease was produced by Po2, which corresponded with the growth and reached a maximum level (264 U/ml) during the stationary phase at 24 h. The production thereafter remained nearly static at optimal level till 36 h. Po2 could grow in the range of 0–20% NaCl (w/v) and pH 7–9, optimally at 10% NaCl (w/v) and pH 8. The protease production was salt-dependent and optimum production required 15% NaCl (w/v) and pH 8. Among the organic nitrogen sources, optimum growth and protease production (260 U/ml) were supported by the combination of peptone and yeast extract. However, growth and protease production were highly suppressed by the inorganic nitrogen sources used; with the exception of potassium nitrate, which supported both growth and protease production to limited extent (24 U/ml). Strong inhibition of enzyme production was observed at above 1% glucose (w/v). Wheat flour served as both carbon and nitrogen source supporting growth and protease production. Introduction Microorganisms represent the most common candidates as sources of new enzymes because of their broad bio- chemical diversity, feasibility of mass culture and ease of genetic manipulation. The exploration of the enzymatic potential of extremophiles has just begun and hitherto only a few enzymes have been explored and investigated (Herbert 1992; Eichler 2001; Rozzell 1999). Proteases are among the commercially most viable enzymes and exploration of further novel microbial sources of these enzymes for applications in many industries such as food and leather (Grebeshova et al. 1988) and as detergent additives (Denizci et al. 2004) has attracted considerable attention. Microbial alkaline proteases dominate the worldwide enzyme market, with a two- third share of the detergent industry (Niehaus et al. 1999; Gupta et al. 2002). Nowadays increasing emphasis is being laid on extremophiles for the presence of such enzymes, mainly due to the mechanisms and strategies that help them to function under stressful growth con- ditions (Madern et al. 2000; Margesin & Schinner 2001). Alkaline proteases from extremophiles have been studied extensively in the recent years (Kim et al. 1991; Ferrero et al. 1996; Johnvesly & Naik 2001; Xiao 2001; Joseph et al. 2002; Kanekar et al. 2002; Saeki 2002; Bakhtiar et al. 2003; Beg & Gupta 2003; Ellaiah et al. 2003; Gessesse et al. 2003; Huang et al. 2003; Denizci 2004; Gupta et al. 2005). In comparison, haloalkaliphilic bacteria have been relatively less attended, as only few alkaline proteases are reported from these organisms (Studdert et al. 1997; Stan-Lotter et al. 1999; Gimenez et al. 2000; Studdert et al. 2001; Polosina et al. 2002). Besides the novel catalytic applications, the wide occur- rence of many of such enzymes among the haloalkali- philic bacteria holds ecological significance. The present work has focused on the production of extracellular alkaline protease from a new haloalkaliphilic bacterium Po2 that has 95% similarity with Bacillus pseudofirmus. Materials and methods Organism Haloalkaliphilic bacterium, Po2, was isolated by enrichment culture technique from a seawater sample from Porbandar, in the coastal region of the Gujarat, Western India. The 1 ml of seawater was added to a complex medium (CMB) containing (g/l): glucose, 10; World Journal of Microbiology & Biotechnology (2006) 22: 375–382 Ó Springer 2006 DOI 10.1007/s11274-005-9044-x