American Journal of Microbiology and Biotechnology 2015; 2(6): 82-91 Published online February 2, 2016 (http://www.aascit.org/journal/ajmb) ISSN: 2375-3005 Keywords Cloning, Expression, Purification, Immobilization, Characterization, Glutathione S Transferase Received: July 26, 2015 Revised: August 5, 2015 Accepted: August 7, 2015 Overexpression, Purification, Immobilization and Characterization of Thermophilic Lipase from Burkholderia pseudomallei Magdy M. Youssef 1, 2 1 Department of Chemistry, College of Science, King Faisal University, Hofuf, Saudi Arabia 2 Department of Chemistry, Faculty of Science, Mansoura University, Mansoura, Egypt Email address myoussef@kfu.edu.sa, mmm_youssef@yahoo.com Citation Magdy M. Youssef. Overexpression, Purification, Immobilization and Characterization of Thermophilic Lipase from Burkholderia pseudomallei. American Journal of Microbiology and Biotechnology. Vol. 2, No. 6, 2015, pp. 82-91. Abstract Lipases (EC 3.1.1.3), triacylglycerol hydrolases, are a significant group of biotechnologically applicable enzymes and they find enormous applications in food, detergent and pharmaceutical industries. Lipases are largely produced from microbes, and they play a fundamental role in commercial ventures. A lipase from thermophilic Burkholderia pseudomallei bacterial strain was isolated from Saudi Arabian environment. Based on this strain, a lipase gene encoding 399 amino acids was cloned, and expressed in E. coli BL21 (DE3). The lipase protein fused with glutathione S- transferase was purified to homogeneity 128.2 fold. SDS- PAGE of the purified enzyme revealed it has Mr of 32 kDa. The recombinant lipase was efficiently immobilized in calcium alginate gelatin composites. The optimum temperature for free enzyme highest activity was recorded at 65°C however the immobilized enzyme exhibits the highest activity at 70°C. The immobilized enzyme retains most of its activity and shows high stability for 120 min at 70°C compared to 45 minutes for the native enzyme when incubated at 70°C. The free enzyme has an optimum pH at 7.5 but this optimum pH is shifted to 8.5 for the immobilized enzyme. The free and immobilized lipase catalytic function were enhanced in the presence of 1 mM of Ba ++ , Ca ++ and Na + , but inhibited by 1mM of Ni ++ , Hg ++ , Cu ++ and Co ++ . The free and the immobilized enzyme activities increased in the presence of 5 mM Fe ++ , Co ++ or Li + . 1. Introduction The natural production of fatty acids by the hydrolysis of natural oils and fats is a very important component in the economic utilization of these naturally produced renewable raw materials. A significant number of high value products require fatty acids in their manufactures. These include coatings, adhesives, specially lubricating oils, shampoos and other personal care products. Oils and fats are part of a group of compounds known as fatty esters or triglycerides, and their hydrolysis essentially involves reactions with water to produce valuable free fatty acids and glycerol [1]. Enzymatic hydrolysis of triglycerides may be carried out at ambient conditions. Microorganisms produce different classes of lipolytic enzymes, including lipases (EC 3.1.1.3), which specifically catalyze the hydrolysis of ester linkages of long chain triglycerides at an oil-water interface [2]. Lipases are currently attracting enormous attention because they constitute the most important group of biocatalysts for biotechnological applications [3-5]. Most of the