Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties H.-S. Joo 1 , C.G. Kumar 2 , G.-C. Park 2 , S.R. Paik 2 and C.-S. Chang 2 1 Institute of Medical Science and 2 Department of Biochemistry, College of Medicine, Inha University, Inchon, Korea 2002/349: received 3 September 2002, revised 20 January 2003 and accepted 17 March 2003 ABSTRACT H.-S. JOO, C.G. KUMAR, G.-C. PARK, S.R. PAIK AND C.-S. CHANG. 2003. Aims: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance. Methods and Results: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l )1 ): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K 2 HPO 4 , 4; Na 2 HPO 4 , 1; MgSO 4 7H 2 O, 0 1; Na 2 CO 3 , 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60°C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H 2 O 2 , respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases. Conclusions: Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H 2 O 2 . Significance and Impact of the Study: The alkaline protease from B. clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations. Keywords: alkaline protease, Bacillus clausii, optimization, production. INTRODUCTION It is well known that proteases constitute one of the most important groups of industrial enzymes, as it accounts for at least a quarter of the total global enzyme production (Layman 1986). In recent years, the use of alkaline proteases in a variety of industrial processes like detergents, food, leather and silk has increased remarkably (Kembhavi et al. 1993; Gessesse 1997 2 ; Kumar and Takagi 1999; Kumar and Tiwari 1999). Although a wide range of micro-organisms are known to date to produce proteases, a large proportion of the commercially available alkaline proteases are derived from Bacillus strains because of its ability to secrete large amounts of alkaline proteases having significant proteolytic activity and stability at considerably high pH and temperatures (Jacobs 1995; Ito et al. 1998; Kumar et al. 1998; Yang et al. 2000). Further, these enzymes are compatible and stable to various detergent components and active at washing tem- peratures and pH values. Among them, a facultative alkalophile Bacillus clausii is known to produce a commer- cially important alkaline serine protease, Savinase Ò 3 (Novo- zyme, Novo Nordisk, Bagsvaerd, Denmark), which finds use as detergent additive to remove protein-containing spots from laundry (Christiansen and Nielsen 2002). In general, most of the alkaline proteases applied for industrial purposes face some limitations. First, many of the available alkaline proteases exhibited low activity and stability towards anionic surfactants like SDS and oxidants like hydrogen peroxide, which have been the common ingredients in modern bleach-based detergent formulations. Secondly, around 30–40% of the production cost of industrial enzymes Correspondence to: Chung-Soon Chang, Department of Biochemistry, College of Medicine, Inha University, 7-241 Shinheung-Dong 3 Ga, Chung-Ku, Inchon 400-103, Korea (e-mail: cschang@inha.ac.kr). ª 2003 The Society for Applied Microbiology Journal of Applied Microbiology 2003, 95, 267–272