ORIGINAL ARTICLE Sequential optimization of production of a thermostable and organic solvent tolerant lipase by recombinant Escherichia coli Rubina Nelofer & Ramakrishnan Nagasundara Ramanan & Raja Noor Zaliha Raja Abd Rahman & Mahiran Basri & Arbakariya B. Ariff Received: 31 July 2010 / Accepted: 19 November 2010 / Published online: 9 December 2010 # Springer-Verlag and the University of Milan 2010 Abstract Several medium formulations were screened for the production of a thermostable and organic solvent tolerant lipase by a recombinant Escherichia coli BL21. The highest lipase production (28.9±4.1 IU/mL) was obtained in Luria Bertani medium with the addition of 1% (w/v) glucose. The medium formulation and fermentation conditions were then subjected to sequential optimization. Using a Plackett-Burman design, glucose, NaCl, tempera- ture and induction time were found to be the most significant variables affecting lipase production, and these were then optimized using response surface methodology (RSM). The large value of R 2 (0.979) showed that the quadratic model used for the prediction is highly signifi- cant. The optimum levels of these four significant variables (glucose, NaCl, temperature and induction time) as pre- dicted by RSM were 32.4 g/L, 5 g/L, 31.7°C and 2.1 h, respectively. The amount of lipase activity (50.2±4.5 IU/ mL) produced under these optimal conditions fitted well to the value (48.9 IU/mL) predicted by RSM. Production of lipase in optimized fermentation was about 2.5-fold higher than in non-optimized fermentation. Keywords Thermostable lipase . Solvent tolerant lipase . Escherichia coli . Fermentation . Optimization . Response surface methodology Introduction Lipases (triacylglycerile hydrolases; EC 3.1.1.3) are industri- ally important enzymes due to their regio-, stereo-, chemo- selective reactions and kinetic resolution of racemates. Conventionally, lipases are used in the detergent industry, as flavour enhancers in the food industry, as fat remover in the meat industry, and as pitch remover in the paper and pulp industry (Hasan et al. 2006). Recently, these enzymes have been used for biodiesel production (Adamczak et al. 2009), enantioselective deacetylation (Kumar and Gupta 2008), cyclic resolution of racemic ibuprofen (Liu et al. 2009), production of medium-chain triacylglycerols (Low et al. 2007) and the preparation of diacylglycerol-enriched palm olein (Wang et al. 2009). Thermostability, organic solvent tolerance and stability over a wide range of pH are the most desired characteristics of industrial lipases. Industrially, lipases are produced in large quantities from microbial fermentation. Lipase-producing strains are isolat- ed from various sources such as oil mill effluent, hot springs and fatty waste (Eltaweel et al. 2005; Rahman et al. 2007). Various microorganisms, such as Staphylococcus caseolyticus (Volpato et al. 2008), Burkholderia multi- vorans (Dandavate et al. 2009), Aureobasidium pullulans (Liu et al. 2008) and Candida cylindracea (Brozzoli et al. 2009), have been identified as lipase producers. Natural R. Nelofer : A. B. Ariff (*) Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia e-mail: arbarif@biotech.upm.edu.my R. N. Ramanan : R. N. Z. R. A. Rahman : M. Basri : A. B. Ariff Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia R. N. Z. R. A. Rahman Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia M. Basri Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia Ann Microbiol (2011) 61:535–544 DOI 10.1007/s13213-010-0170-9