Production of a Pseudomonas lipase in n-alkane substrate and its isolation using an improved ammonium sulfate precipitation technique Lambit Kanwar, Binod Kumar Gogoi, Pranab Goswami * Biochemistry Division, Regional Research Laboratory, Jorhat 785 006, Assam, India Received 10 April 2001; received in revised form 22 February 2002; accepted 7 March 2002 Abstract Among the various lipidic and non-lipidic substances, normal alkanes within the chain lengths of C-12 to C-20 served as the best carbon substrates for the production of extracellular lipase by Pseudomonas species G6. Maximum lipase production of 25 U/ml of the culture broth was obtained by using n-hexadecane as the sole carbon substrate. The optimum pH of 8 and temperature of 34  1 °C were demonstrated for the production of lipase in n-hexadecane substrate. The optimum concentration of iron, which played a critical role on the lipase production, was found to be 0.25 mg/l. Lipase production could be enhanced to nearly 2.4-fold by using tributyrin at a concentration of 0.05% (v/v) in the culture medium. High recovery of the lipase protein (83%) from the culture broth was achieved by treating the culture supernatant with Silicone 21 Defoamer followed by ammonium sulfate (60% saturation) fractionation. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Pseudomonas; Lipase; n-Alkane 1. Introduction The cost of microbial lipase enzymes is largely de- termined by the yield which is related to the amount of enzyme produced, down stream processing and post harvest stability. Among the various factors that influ- ence lipase production during culture, the type of carbon substrates and inducer have a profound effect on the production of microbial lipases. Because microbial lip- ases function to break down insoluble lipidic substrates that can be more easily absorbed, most microbial lipases are produced extracellularly (Saxena et al., 1999). However, many microbial strains secrete lipase to the medium during their growth on non-lipidic substrates such as amino acids, sugars and other organic com- pounds (Lakshmanan, 1991; Makhzoum et al., 1995). The petroleum hydrocarbons, which are also non-lipidic compounds, have been reported as being possible sub- strates for the microbial production of extracellular li- pase though the study is limited to a very few fungal strains (Takahashi et al., 1963; Chen et al., 1994). The successful exploitation of enzymes depends gen- erally on the development of efficient and reliable methods for their separation and purification. Different techniques have been used for the purification of en- zymes (Jakoby, 1984). The ammonium sulfate precipi- tation of lipase and other enzymes is the commonly used technique because of its low cost, high solubility and its protective nature on the enzymes. However, application of some other techniques, such as, foam flotation and miceller extraction using surfactants, is also gaining importance in recent years (Sarada, 1994; Hayes and Marchio, 1998; Sch€ ugerl, 2000). In this paper we report effects of n-alkanes on lipase production by a soil isolate Pseudomonas species G6. An improved ammonium sulfate precipitation technique for the isolation of the lipase enzyme from the culture broth is also described. 2. Methods 2.1. Organism and culture condition The Pseudomonas species G6 used in this study was isolated from the crude oil contaminated soil sample by Bioresource Technology 84 (2002) 207–211 * Corresponding author. Fax: +91-376-33-70011. E-mail address: pranab_goswami@yahoo.com (P. Goswami). 0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII:S0960-8524(02)00061-5