Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast Pooja Shivanand, Gurunathan Jayaraman * School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore 632014, India 1. Introduction Proteases constitute one of the most important groups of industrial enzymes with versatile applications including meat tenderization, detergents, cheese-making, de-hairing, baking, waste management and silver recovery [1,2]. They are involved in a multitude of physiological reactions and cell signaling systems. Recently, there has been an increased interest in proteases as targets for developing therapeutic agents [3]. According to the market research report on world enzymes published in 2007, the world market for enzymes is expected to grow 7.6% per year to $6 billion in 2011 [4]. Microbial proteases account for approximately 60% of the total enzyme sales in the world [5]. One of the several challenges faced by industrial application of microbial proteases is optimal activity and stability in a wide range of salinity. Halophilic proteases are less suitable for saline fermentation processes, because they need at least 12.5% (w/v) NaCl for expression of high activities [6]. Therefore, halotolerant proteases, which are active both at low and high concentrations of NaCl, are needed. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production [7]. Halophiles are microorganisms which grow over an extended range of salt concentrations (3–30% NaCl, w/v) and include the halotolerant bacteria and the obligate halophilic archaea. They are found in salt marshes, marine ecosystems, salted meats, hypersa- line seas, salt evaporation pools and salt mines [6]. Halotolerant bacteria form a versatile group; adapted to life at the lower range of salinities, with the possibility of rapid adjustment to changes in the external salt concentration. In contrast, the halophilic archaea are strictly dependent on the constant presence of high salt concentrations (3–4 M) for survival [8]. This property of haloto- lerant bacteria makes them better candidates for bio-prospecting than their halophilic counterparts. While most of the reports on halotolerant bacteria focus on molecular phylogeny, only a few reports are available on designing bioprocesses that improve growth conditions, and then positively influence the productivity of biomass, enzymes or metabolites [9]. Most of the Gram-positive or Gram-variable, endospore- forming rods with halotolerant properties, have been assigned to the genus Bacillus [10]. Bacillus sp. grows in a pH range of 7.0– 11.0 and produces extracellular protease. Currently, a large proportion of commercially available alkaline proteases are derived from different strains of Bacillus [11,12]. Most aquatic microbes are halotolerant and are reported to synthesise exozymes Process Biochemistry 44 (2009) 1088–1094 ARTICLE INFO Article history: Received 25 February 2009 Received in revised form 19 May 2009 Accepted 21 May 2009 Keywords: Bacillus aquimaris Protease production Halotolerant bacteria Extremophiles Salt tolerance Saltern ABSTRACT A newly isolated halotolerant Bacillus sp. VITP4 was investigated for the production of extracellular protease. 16S rRNA gene analysis identified it as Bacillus aquimaris. Enzyme secretion corresponded with growth (G t , 38 min) in the basal Zobell medium, reaching a maximum during stationary phase (630 U/ml, 48 h). Protease production was investigated in different salt concentrations (0–4 M). While growth was optimum in the basal medium, higher levels of protease activity were observed in 0.5 M salt medium (728 U/ml, 48 h) and 1 M salt medium (796 U/ml, 78 h) with 21% and 32% increase in production, respectively. Salt concentrations above 2.5 M did not support bacterial growth. The optimum pH and temperature for production were pH 7.5 and 37 8C, respectively. A combination of peptone and yeast extract yielded optimum protease secretion. Inorganic nitrogen sources proved to be less favourable. Production was reduced in the presence of readily available carbon sources owing to catabolic repression. Effect of various salts (1–6%) indicated favourable bacterial growth in these conditions for producing proteolytic molecules with increased activity. The study assumes significance in the ability of the halotolerant bacterium to survive in a wide range of salinity and yield optimum levels of extracellular protease. ß 2009 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +91 416 2202573; fax: +91 416 2243092. E-mail address: gjayaraman@vit.ac.in (G. Jayaraman). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.05.010