Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: Compatibility study with detergent ingredients and washing performance Sanja Grbavc ˇic ´ a , Dejan Bezbradica b , Lidija Izrael-Z ˇ ivkovic ´ c , Nataša Avramovic ´ c , Nenad Milosavic ´ d , Ivanka Karadz ˇic ´ c , Zorica Knez ˇevic ´ -Jugovic ´ b,⇑ a Innovation Center, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11001 Belgrade, Serbia b Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11001 Belgrade, Serbia c Department of Chemistry, School of Medicine, University of Belgrade, Visegradska 26, 11000 Belgrade, Serbia d Department of Biochemistry, Faculty of Chemistry, Studentski trg 12-16, 11001 Belgrade, Serbia article info Article history: Received 1 June 2011 Received in revised form 19 September 2011 Accepted 20 September 2011 Available online 29 September 2011 Keywords: Pseudomonas aeruginosa Lipase Protease Detergent Medium optimization abstract An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compat- ibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol Ò XP80 and Triton Ò X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1 h) while the protease activity was enhanced by the addition of Triton Ò WR1339 and Tween Ò 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Contemporary cleaning products are composite mixtures of several active ingredients differing on the purpose of the detergent. Apart from surfactants as main components, detergents contain builders, corrosion inhibitors, optical brighteners, foam regulators, bleaching agents and enzymes among other auxiliary additives. Detergent performance and cleaning efficiency are results of a combination of factors that act synergically on the soiled material (Jurado et al., 2007). Proteases and lipases are the key enzymatic constituents in detergent formulations. The functional importance of lipases and proteases in the detergent industry is that of improving the wash- ing capacity of detergents and removing fatty and proteinaceous food stains in laundry, which are difficult to remove under mild washing condition (Jurado et al., 2007; Saeki et al., 2007; Gaur et al., 2008a). Besides the removal of fatty residues in laundry and dishwashers, lipases can be used in formulations for cleaning of clogged drains (Bora and Kalita, 2008; Rathi et al., 2001). Detergent formulations offer several particularly hard chal- lenges for an enzyme. Washing formulations containing enzymes often operate at elevated temperatures of 30–60 °C, in alkaline pH values of 9–12 in the presence of inhibitory or deactivating compounds. Not all lipases and proteases with a potential for fat and protein-based stain degradation and/or removal can actually be effective as detergent ingredients. The enzyme must be compat- ible with detergent components and have a good performance at relevant washing temperature and (washing) pH. Furthermore, their substrate specificity should be low. The most important detergent lipase presently used is that from Thermomyces sp., expressed in recombinant strains of Aspergillus oryzae (Lipolase Ò , Novozymes) (Gillis, 1988). Other formulations such as Lumafast Ò and Lipomax Ò (Gist-Brocades) contain Pseudo- monas spp. lipases (Lenting et al., 1993). These lipases have the rather unconventional properties required (good stability and opti- mal activity at alkaline pH and temperatures of 40 °C or higher), making them attractive choices for detergent application. Thus, a large number of Pseudomonas species have been studied for lipase production (Makhzoum et al., 1995; Lin, 1996; Ito et al., 2001; Gaur et al., 2008a,b). Significant commercial detergent protease additives such as Savinase Ò , Esperase Ò , Alcalase Ò (Novozymes), Maxacal Ò , Maxinase Ò (Gist-Brocades) are mostly produced by Bacillus spp. (Gupta et al., 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.09.076 ⇑ Corresponding author. Tel.: +381 (11) 3303776; fax: +381 (11) 3370 387. E-mail address: zknez@tmf.bg.ac.rs (Z. Knez ˇevic ´ -Jugovic ´). Bioresource Technology 102 (2011) 11226–11233 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech