A novel surfactant-stable alkaline serine-protease from a newly isolated Bacillus mojavensis A21. Purification and characterization Anissa Haddar, Ali Bougatef, Rym Agrebi, Alya Sellami-Kamoun, Moncef Nasri * Laboratoire de Ge ´nie Enzymatique et de Microbiologie, Ecole Nationale d’Inge ´nieurs de Sfax, B.P. ‘‘W’’, 3038 Sfax, Tunisia 1. Introduction Microbial proteases constitute approximately 40% of the total worldwide production of enzymes [1]. They have diverse applications in a wide variety of industries, such as in detergent, food, pharmaceutical, leather, silk and for recovery of silver from used X-ray films [2,3]. Of these, alkaline proteases are particu- larly important because they are both stable and active at high pH solutions and in the presence of surfactants and oxidizing agents [3,4]. Their major application is in detergent industry, because the pH of laundry detergents is generally in the range of 9.0–12.0. Alkaline proteases are used as cleaning additives in detergents to facilitate the release of proteins. Proteases in detergent industries account for approximately 30% of the total world enzyme production [5]. They are produced by a wide range of microorganisms including bacteria, moulds, yeasts and also mammalian tissues. Most of the commercial alkaline proteases were isolated from Bacillus species [6]. The industrial demand of highly active preparations of proteolytic enzymes with appropriate specificity and stability to pH, temperature, metal ions and surfactants continues to stimulate the search for new enzyme sources. Isolation and screening of microorganisms from naturally occurring alkaline habitats or from alkaline wastewater is expected to provide new strains producing enzymes active and stable in high alkaline conditions. Many alkaline proteases have been purified and characterized [7–10]. Subtilisin Carlsberg produced by B. licheniformis [8] and Subtilisin Novo produced by B. amyloliquefaciens [9] have been the enzymes of choice in detergent formulations. Both subtilisins have a molecular mass of 27.5 kDa but differ from each other by 58 amino acids. These enzymes showed maximum activity at pH values of 8.0–10.0 [10]. The optimum pH and temperature for the proteolytic activity of the purified protease from Aspergillus clavatus ES1 were pH 8.5 and 50 8C [11]. Process Biochemistry 44 (2009) 29–35 ARTICLE INFO Article history: Received 15 April 2008 Received in revised form 26 August 2008 Accepted 9 September 2008 Keywords: Alkaline protease Surfactant-stable Bacillus mojavensis Purification ABSTRACT An extracellular bleach stable protease producing strain was isolated from marine water sample and identified as Bacillus mojavensis A21 on the basis of the 16S rRNA gene sequencing and biochemical properties. The A21 alkaline protease was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration and CM-Sepharose ion exchange chromatography, with a 6.43-fold increase in specific activity and 16.56% recovery. The molecular weight of the purified enzyme was estimated to be 20 kDa by SDS-PAGE and gel filtration. The enzyme was highly active over a wide range of pH from 7.0 to 13.0, with an optimum at pH 8.5. The relative activities at pH 11.0 and 12.0 were about 80 and 71.7% of that obtained at pH 8.5. The enzyme was extremely stable in the pH range of 7.0–12.0. It exhibited maximal activity at 60 8C. The thermostability of the enzyme was significantly increased by the addition of CaCl 2 . The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The N-terminal amino acid sequence of the first 20 amino acids of the purified protease was DINGGGATLPQKLYQTSGVL. B. mojavensis A21 protease showed low homology with bacterial peptidases, suggesting that the enzyme is a new protease. The alkaline protease showed high stability towards anionic (0.1% SDS) and non-ionic (1 and 5% Tween 80 and 1% Triton X-100) surfactants. In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 79 and 70% of its initial activity after 1 h incubation in the presence of 1% H 2 O 2 and 0.1% sodium perborate, respectively. The enzyme showed excellent stability with a wide range of commercial solid and liquid detergents at 30 and 40 8C. Considering its promising properties, B. mojavensis A21 may find potential application in laundry detergents. ß 2008 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +216 74 274 088; fax: +216 74 275 595. E-mail addresses: mon_nasri@yahoo.fr, moncef.nasri@enis.rnu.tn (M. Nasri). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.09.003