Characterization of a Thermostable, Recombinant Carboxylesterase from the Hyperthermophilic Archaeon Metallosphaera sedula DSM5348


Lipid-producing microalgae are emerging as the leading platform for producing alternative biofuels in response to diminishing petroleum reserves. Optimization of fatty acid production is required for efficient conversion of microalgal fatty acids into usable transportation fuels. Microbial lipases/esterases can be used to enhance fatty acid production because of their efficacy in catalyzing hydrolysis of esters into alcohols and fatty acids while minimizing the potential poisoning of catalysts needed in the biofuel production process. Although studies have extensively focused on lipases/esterases produced by mesophilic organisms, an understanding of lipases/esterases produced by thermophilic, acidic tolerant microbes, such as Metallosphaera sedula, is limited. In this work, the carboxylesterase from Metallosphaera sedula DSM5348 encoded by Msed_1072 was recombinantly expressed in Escherichia coli strain BL21 (λDE3). The purified enzyme either with a hexahistidine (His6)-tag (Msed_1072Nt and Msed_1072Ct) or without the hexahistidine (His6)-tag (Msed_1072) was biochemically characterized using a variety of substrates over a range of temperatures and pH and in the presence of metal ions, organic solvents, and detergents. In this study, the fusion of the protein with a hexahistidine (His6)-tag did not result in a change in substrate specificity, but the findings provide information on which enzyme variant can hydrolyze fatty acid esters in the presence of various chemicals, and this has important implication for their use in industrial processes. It also demonstrates that Metallosphaera sedula Msed_1072 can have application in microalgae-based biofuel production systems.

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Killens-Cade, R. , Turner, R. , MacInnes, C. and Grunden, A. (2014) Characterization of a Thermostable, Recombinant Carboxylesterase from the Hyperthermophilic Archaeon Metallosphaera sedula DSM5348. Advances in Enzyme Research, 2, 1-13. doi: 10.4236/aer.2014.21001.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Jaeger, K.E. and Eggert, T. (2002) Lipases for Biotechnology. Current Opinion in Biotechnology, 13, 390-397.
[2] Gilham, D. and Lehner, R. (2005) Techniques to Measure Lipase and Esterase Activity in Vitro. Methods, 36, 139-147.
[3] Jaeger, K.E., Dijkstra, B.W. and Reetz, M.T. (1999) Bacterial Biocatalysts: Molecular Biology, Three-Dimensional Structures, and Biotechnological Applications of Lipases. Annual Review of Microbiology, 53, 315-351.
[4] Panda, T. and Gowrishankar, B.S. (2005) Production and Applications of Esterases. Applied Microbiology and Biotechnology, 67, 160-169.
[5] Salameh, M.A. and Wiegel, J. (2007) Purification and Characterization of Two Highly Thermophilic Alkaline Lipases from Thermosyntropha lipolytica. Applied and Environmental Microbiology, 73, 7725-7731.
[6] Menetrez, M.Y. (2012) An Overview of Algae Biofuel Production and Potential Environmental Impact. Environmental Science and Technology, 46, 7073-7085.
[7] Pienkos, P.T. and Darzins, A. (2009) The Promise and Challenges of Microalgal-Derived Biofuels. Biofuels, Bioproducts, & Biorefining, 3, 431-440.
[8] Guschina, I.A. and Harwood, J.L. (2006) Lipids and Lipid Metabolism in Eukaryotic Algae. Progress in Lipid Research, 45, 160-186.
[9] Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M. and Darzins, A. (2008) Microalgal Triacylglycerols as Feedstocks for Biofuel Production: Perspectives and Advances. Plant Journal, 54, 621-639.
[10] Liu, X., Sheng, J. and Curtiss, R. (2010) Fatty Acid Production in Genetically Modified Cyanobacteria. Proceedings of the National Academy of Sciences, 108, 6899-6904.
[11] Voelker, T.A. and Davies, H. (1994) Alteration of the Specificity and Regulation of Fatty Acid Synthesis of Escherichia coli by Expression of a Plant Medium-Chain Acyl-Acyl Carrier Protein Thioesterase. Journal of Bacteriology, 176, 7320-7327.
[12] Barnes, E.M. and Wakil, S.J. (1968) Studies on the Mechanism of Fatty Acid Synthesis. XIX. Preparation and General Properties of Palmityl Thioesterase. Journal of Biological Chemistry, 243, 2955-2962.
[13] Huber, G., Spinnler, C., Gambacorta, A. and Stetter, K.O. (1989) Metallosphaera sedula gen. and sp. nov. Represents a New Genus of Aerobic, Metal-Mobilizing, Thermoacidophilic Archaebacteria. Systematic and Applied Microbiology, 12, 38-47.
[14] Lee, Y.L., Su, M.S., Huang, T.H. and Shaw, J.F. (1999) C-Terminal His-Tagging Results in Substrate Specificity Changes of the Thioesterase I from Escherichia coli. Journal of the American Oil Chemists’ Society, 76, 1113-1118.
[15] Studier, F. (2005) Protein Production by Auto-Induction in High Density Shaking Cultures. Protein Expression and Purification, 41, 207-234.
[16] Bannon, C.D., Craske, J.D., Hai, N.T., Hai, N.L., Happer, N.L. and O’Rourke, K.L. (1982) Analysis of Fatty Acid Methyl esters with High Accuracy and Reliability : II. Methylation of Fats and Oils with Boron Trifluoride-Methanol. Journal of Chromatography A, 247, 63-69.
[17] Sparkman, O.D., Penton, Z.E. and Kitson, F.G. (2011) Gas Chromatography and Mass Spectrometry: A Practical Guide. 2nd Edition, Elsevier, Inc., Burlington.
[18] Liu, L.J., You, X.Y., Zheng, H., Wang, S., Jiang, C.Y. and Liu, S.J. (2011) Complete Genome Sequence of Metallosphaera cuprina, a Metal Sulfide-Oxidizing Archaeon from a Hot Spring. Journal of Bacteriology, 193, 3387-3388.
[19] Reno, M.L., Held, N.L., Fields, C.J., Burke, P.V. and Whitaker, R.J. (2009) Biogeography of the Sulfolobus islandicus Pan-Genome. Proceedings of the National Academy of Sciences, 106, 8605-8610.
[20] She, Q., Singh, R.K., Confalonieri, F., Zivanovic, Y., Allard, G., Awayez, M.J., Chan-Weiher, C.C., Clausen, I.G., Curtis, B.A., De Moors, A., Erauso, G., Fletcher, C., Gordon, P.M., Heikamp, D.E., Jong, I., Jeffries, A.C., Kozera, C.J., Medina, N., Peng, X., Thi-Ngoc, H.P., Redder, P., Schenk, M.E., Theriault, C., Tolstrup, N., Charlebois, R.L., Doolittle, W.F., Duguet, M., Gaasterland, T., Garrett, R.A., Ragan, M.A., Sensen, C.W. and van der Oost, J. (2001) The Complete Genome of the Crenarchaeon Sulfolobus solfataricus P2. Proceedings of the National Academy of Sciences, 98, 7835-7840.
[21] Jaeger, K.E., Ransac, S., Dijkstra, B.W., Colson, C., van Heuvel, M. and Misset, O. (1994) Bacterial Lipases. FEMS Microbiology Reviews, 15, 29-63.
[22] De Simone, G., Menchise, V., Manco, G., Mandrich, L., Sorrentino, N., Lang, D., Rossi, M. and Pedone, C. (2001) The Crystal Structure of a Hyper-Thermophilic Carboxylesterase from the Archaeon Archaeoglobus fulgidus. Journal of Molecular Biology, 314, 507-518.
[23] Byun, J.S., Rhee, J.K., Kim, D.U., Oh, J.W. and Cho, H.S. (2006) Crystallization and Preliminary X-Ray Crystallographic Analysis of EstE1, a New and Thermostable Esterase Cloned from a Metagenomic Library. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 62, 145-147.
[24] Angkawidjaja, C., Koga, Y., Takano, K., and Kanaya, S. (2012) Structure and Stability of a Thermostable Carboxylesterase from the Thermoacidophilic Archaeon Sulfolobus tokodaii. FEMS Microbiology Letters, 276, 3071-3084. 10.1111/j.1742-4658.2012.08687.x
[25] Levisson, M., Sun, L., Hendriks, S., Swinkels, P., Akveld, T., Bultema, J., Barendregt, A., van den Heuvel, R., Dijkstra, B., ver der Oost, J. and Kengen, S. (2009) Crystal Structure and Biochemical Properties of a Novel Thermostable Esterase Containing an Immunoglobulin-Like Domain. Journal of Molecular Biology, 385, 949-962.
[26] Atomi, H., Fukui, T., Kanai, T., Morikawa, M. and Imanaka, T. (2004) Description of Thermococcus kodakaraensis sp. nov., a Well Studied Hyperthermophilic Archaeon Previously Reported as Pyrococcus sp. KOD1. Archaea, 1, 263-267.
[27] Levisson, M., van der Oost, J. and Kengen, S.W. (2009) Carboxylic Ester Hydrolases from Hyperthermophiles. Extremophiles, 13, 567-581.
[28] Sehgal, A.C., Callen, W., Mathur, E.J., Short, J.M. and Kelly, R.M. (2001) Carboxylesterase from Sulfolobus solfataricus P1. Methods in Enzymology, 330, 461-471.
[29] Harwood, J. (1998). Membrane lipids in algae. In Lipids in Photosynthesis: Structure, Function, and Genetics. Kluwer Academic Publishers, Dordrecht, 53-64.
[30] Mandrich, L., Merone, L., Pezzullo, M., Cipolla, L., Nicotra, F., Rossi, M. and Manco, G. (2005) Role of the N Terminus in Enzyme Activity, Stability and Specificity in Thermophilic Esterases Belonging to the HSL Family. Journal of Molecular Biology, 345, 501-512.
[31] Matsunaga, A., Koyama, N. and Noso, Y. (1974) Purification and Properties of Esterase from Bacillus stearothermophilus. Archives of Biochemistry and Biophysics, 160, 504-513.
[32] Jaeger, K.E. and Reetz, M.T. (1998) Microbial Lipases form Versatile Tools for Biotechnology. Trends in Biotechnology, 16, 396-403.
[33] Han, S.J., Back, J.H., Yoon, M.Y., Shin, P.K., Cheong, C.S., Sung, M.H., Hong, S.P., Chung, I.Y. and Han, Y.S. (2003) Expression and Characterization of a Novel Enantioselective Lipase from Acinetobacter Species SY-01. Biochimie, 85, 501-510.
[34] Manco, G., Mandrich, L. and Rossi, M. (2001) Residues at the Active Site of the Esterase 2 from Alicyclobacillus acidocaldarius Involved in Substrate Specificity and Catalytic Activity at High Temperature. Journal of Biological Chemistry, 276, 37482-37490.

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