International Journal of Science and Technology Volume 2 No. 7, July, 2013 IJST © 2013 – IJST Publications UK. All rights reserved. 502 Screening and Molecular Characterization of Extracellular Lipase Producing Bacillus Species from Coconut Oil Mill Soil V.R. Gayathri 1 , P. Perumal 1 , Lini P Mathew 2 ., B. Prakash 3 1 Department of Biotechnology, Periyar University, Salem, Tamilnadu, India 2 Acme Progen Biotech India Private Limited, Salem, Tamilnadu, India 3 Department of Biotechnology, PGP College of Arts & Science, Namakkal, Tamilnadu, India ABSTRACT Lipases (triacylglycerol acylhydrolases E.C 3.1.1.3) are ubiquitous enzymes of considerable physiological significance and industrial application. In view of this significance of lipase we performed screening and molecular characterization of extracellular lipase producing Bacillus species in soil samples collected from coconut oil mill. The dominant Lipase producing organisms were isolated from oil-spilled areas of the groundnut oil extracting industry at Salem. The isolates were identified as Bacillus spp, the influence of growth period on lipase production of Bacillus spp. (B1 – B5) was assessed by culturing it on media for 48hrs. Further, the influence of substrate concentration (2 to 4 %) on lipase production was also assessed on the optimized substrate, which maximized the lipase production. Isolated Bacillus spp.(B1 – B5) were assayed for extra cellular lipase production. One unit of lipase activity was defined as the amount of enzyme releasing one mole of free fatty acid in one minute under standard assay condition. The purification method consists of Ammonium Sulphate precipitation, Dialysis and Column chromatography and Molecular determination by SDS PAGE. In genotypic characterization, RAPD analysis was done to characterize the organism. Keywords: Lipase, Bacillus spp., SDS PAGE and RAPD. 1. INTRODUCTION Lipases are important enzyme with significant commercial applications in industries. Lipases catalyze the hydrolysis of triacyl glycerols to glycerol and free fatty acids, in contrast to esterases. Lipases are activated only when adsorbed to an oil-water interface and do not hydrolyze dissolved substrates in the bulk fluid. A true lipase will split emulsified esters of glycerine and long- chain fatty acids such as triolein and tripalmitin. Among the various factors that influence lipase production during culture, the type of carbon substrates and inducers, have a profound effect on the production of microbial lipases because microbial lipases function is to break down insoluble lipidic substrates so that they can be more easily absorbed (Saxena et al., 1999). Lipases find promising applications in organic chemical processing, detergent formulations, synthesis of biosurfactants, the chemical and paper industries, nutrition, cosmetics, and pharmaceutical processing. Development of lipase-based technologies for the synthesis of novel compounds is rapidly expanding the uses of these enzymes. Increased productivity of lipase during the fermentation process is of great importance, and lower cost of production could promote new industrial applications. However, interest in bacterial lipases has increased because they are more stable than those from other organisms, especially when exposed to high temperatures and other severe conditions (Sugihara et al., 1991). Recently, there has been considerable interest in the basic properties and industrial applications of thermostable lipases from mesophiles and thermophiles. The present study is to investigate the extracellular lipase producing Bacillus Species isolated from coconut oil mill soil. 2. MATERIALS AND METHODS Soil sample were aseptically collected from oil-spilled areas of the oil extracting industry. Both dilution plate and enrichment method were used for the initial screening and isolation of bacterial species. For the enrichment method, 1g of samples were subjected to heat treatment for 10 min at 80ºC in a water bath in order to kill most of the vegetative cells and thus to eliminate non-spore forming bacteria (Moraet al., 1998). After heat treatment, the samples were transferred into 100 ml of Tributyrin medium. Incubation was performed in a rotary shaker at 50 0 C until turbidity obtained. Then 500 μl of the broth was plated on Tributyrin medium. For the dilution plate method 1g of samples were transferred in 9 ml of 0.85% saline water. After pasteurization at 80 0 C for 10 min, 1 ml aliquot from each of the samples was transferred in 9 ml of 0.85% saline water and 6 fold dilutions were prepared. One ml of dilution was plated on Tributyrin medium plates and incubated for 48-72 h at 37 0 C. Single colonies with different morphologies were picked and purified using streak plate method. Screening for Lipase Activity The media described were used in lipase screening. After inoculation of the isolates, the plates were incubated for