Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v); yeast extract 1.25% (w/v); NaCl 0.45% (w/v) olive oil 0.1% (v/v) with agitation of 200？rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30？kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16) and olive oil with optimal activity (100%) compared to other substrates. 1. Introduction Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyse the hydrolysis of long-chain triglycerides with the formation of diacylglycerol, monoacylglycerol, glycerol, and carboxylate, as well as the reverse reaction of the synthesis of esters formed from fatty acids and glycerols [1], present in diverse organisms including animals, plants, fungi, and bacteria. However, only microbial thermostable lipases are commercially significant for their potential use in industries, such as specialty organic syntheses [2], hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, and chemical analyses [3]. Microbial lipases also have been immensely used for biotechnological applications in dairy, detergents, and textile industries as well as surfactant and oil-processing industries. In fact they have also been widely used in pharmaceutical industries in the production of enaniometrically pure chemicals, since they have a number of unique characteristics couple with in district substrate specificity [4], stable and active in organic solvents [5], do not require cofactors [6], exhibit a high degree of regioselectivity, and possess a wide range of substrate specificity for the conversion of various unnatural substrates [2]. Lipases with molecular weight range of 19–60？kDa, belong to the α/β hydrolase family. The active site is formed by a catalytic triad of Ser, Asp/Glu and His [7].
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