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 126.96.36.199) 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 , 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 , hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, and chemical analyses . 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 , stable and active in organic solvents , do not require cofactors , exhibit a high degree of regioselectivity, and possess a wide range of substrate specificity for the conversion of various unnatural substrates . 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 .
I. Kauffmann and C. Schmidt-Dannert, “Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design,” Protein Engineering, vol. 14, no. 11, pp. 919–928, 2001.
V. Nagy, E. R. Toke, L. C. Keong et al., “Kinetic resolutions with novel, highly enantioselective fungal lipases produced by solid state fermentation,” Journal of Molecular Catalysis B, vol. 39, no. 1–4, pp. 141–148, 2006.
F. Niehaus, C. Bertoldo, M. K？hler, and G. Antranikian, “Extremophiles as a source of novel enzymes for industrial application,” Applied Microbiology and Biotechnology, vol. 51, no. 6, pp. 711–729, 1999.
D. A. Cowan and R. Fernandez-Lafuente, “Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization,” Enzyme and Microbial Technology, vol. 49, no. 4, pp. 326–346, 2011.
C. Ash, J. A. E. Farrow, S. Wallbanks, and M. D. Collins, “Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences,” Letters in Applied Microbiology, vol. 13, no. 4, pp. 202–206, 1991.
T. N. Nazina, T. P. Tourova, A. B. Poltaraus et al., “Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans,” International Journal of Systematic and Evolutionary Microbiology, vol. 51, no. 2, pp. 433–446, 2001.
F. A. Rainey, D. Fritze, and E. Stackebrandt, “The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis,” FEMS Microbiology Letters, vol. 115, no. 2-3, pp. 205–211, 1994.
M. Kambourova, N. Kirilova, R. Mandeva, and A. Derekova, “Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7,” Journal of Molecular Catalysis B, vol. 22, no. 5-6, pp. 307–313, 2003.
A. Balan, N. Magalinggam, D. Ibrahim, and R. A. Rahim, “Thermostable lipase production by Geobacillus thermodenitrificans in a 5-L stirred-tank bioreactor,” Internet Journal of Microbiology, vol. 8, no. 2, 9 pages, 2010.
S. Kumar, K. Kikon, A. Upadhyay, S. S. Kanwar, and R. Gupta, “Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3,” Protein Expression and Purification, vol. 41, no. 1, pp. 38–44, 2005.
C. Sharon, M. Nakazato, H. I. Ogawa, and Y. Kato, “Lipase-induced hydrolysis of castor oil: effect of various metals,” Journal of Industrial Microbiology and Biotechnology, vol. 21, no. 6, pp. 292–295, 1998.
M. Sifour, H. M. Saeed, T. I. Zaghloul, M. M. Berekaa, and Y. R. Abdel-Fattah, “Purification and properties of a lipase from thermophilic Geobadllus stearothermophilus strain-5,” International Journal of Biological Chemistry, vol. 4, no. 4, pp. 203–212, 2010.
M. Chartrain, L. Katz, C. Marcin et al., “Purification and characterization of a novel bioconverting lipase from Pseudomonas aeruginosa MB 5001,” Enzyme and Microbial Technology, vol. 15, no. 7, pp. 575–580, 1993.
M. Kohno, W. Kugimiya, Y. Hashimoto, and Y. Morita, “Purification, characterization, and crystallization of two types of lipase from Rhizopus niveus,” Bioscience, Biotechnology and Biochemistry, vol. 58, no. 6, pp. 1007–1012, 1994.
K. Ohnishi, Y. Yoshida, J. Toita, and J. Sekiguchi, “Purification and characterization of a novel lipolytic enzyme from Aspergillus oryzae,” Journal of Fermentation and Bioengineering, vol. 78, no. 6, pp. 413–419, 1994.
R. Sharma, S. K. Soni, R. M. Vohra, L. K. Gupta, and J. K. Gupta, “Purification and characterisation of a thermostable alkaline lipase from a new thermophilic Bacillus sp. RSJ-1,” Process Biochemistry, vol. 37, no. 10, pp. 1075–1084, 2002.
S. S. Kanwar, I. A. Ghazi, S. S. Chimni et al., “Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate,” Protein Expression and Purification, vol. 46, no. 2, pp. 421–428, 2006.
C. Schmidt-Dannert, M. L. Rúa, and R. D. Schmid, “Two novel lipases from thermophile Bacillus thermocatenulatus: screening, purification, cloning, overexpression, and properties,” Methods in Enzymology, vol. 284, pp. 194–220, 1997.
H. K. Kim, S. Y. Park, J. K. Lee, and T. K. Oh, “Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1,” Bioscience, Biotechnology and Biochemistry, vol. 62, no. 1, pp. 66–71, 1998.
J. M. Palomo, R. L. Segura, G. Fernández-Lorente et al., “Purification, immobilization, and stabilization of a lipase from Bacillus thermocatenulatus by interfacial adsorption on hydrophobic supports,” Biotechnology Progress, vol. 20, no. 2, pp. 630–635, 2004.
Y. Wang, K. C. Srivastava, G. J. Shen, and H. Y. Wang, “Thermostable alkaline lipase from a newly isolated thermophilic Bacillus, strain A30-1 (ATCC 53841),” Journal of Fermentation and Bioengineering, vol. 79, no. 5, pp. 433–438, 1995.
R. N. Z. R. A. Rahman, S. N. Baharum, M. Basri, and A. B. Salleh, “High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5,” Analytical Biochemistry, vol. 341, no. 2, pp. 267–274, 2005.
N. S. Abdul Hamid, H. B. Zen, O. B. Tein, Y. M. Halifah, N. Saari, and F. Abu Bakar, “Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring,” World Journal of Microbiology and Biotechnology, vol. 19, no. 9, pp. 961–968, 2003.