Abstract
Enzymes isolated from extremophiles often exhibit superior performance and potential industrial applications. There are several advantages performing biocatalysis at elevated temperatures, including enhanced reaction rates, increased substrate solubility and decreased risks of contamination. Furthermore, thermophilic enzymes usually exhibit high resistance against many organic solvents and detergents, and are also more resistant to proteolytic attack. In this study, we subcloned and characterized an esterase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf_Est) that exhibits optimal activity around 80 °C using naphthol-derived substrates and p-nitrophenyl palmitate (pNPP). According to the circular dichroism spectra, the secondary structure of P. furiosus esterase, which is predominantly formed by a β-sheet structure, is very stable, even after incubation at 120°C. We performed SAXS to determine the low-resolution structure of Pf_Est, which is monomeric in solution at 80 °C and has a molecular weight of 28 kDa. The Km and V max values for this esterase acting on pNPP were 0.53 mmol/L and 6.5 × 10−3 U, respectively. Pf_Est was most active in the immiscible solvents and retained more than 50 % in miscible solvents. Moreover, Pf_Est possesses transesterification capacity, presenting better results when isobutanol was used as an acyl acceptor (2.69 ± 0.14 × 10−2 μmol/min mg) and the highest hydrolytic activity toward olive oil among different types of oils testes in this study. Collectively, these biophysical and catalytic properties are of interest for several biotechnological applications that require harsh conditions, including high temperature and the presence of organic solvents.
Similar content being viewed by others
References
Arpigny, J. L., & Jaeger, K. E. (1999). Bacterial lipolytic enzymes: classification and properties. The Biochemical Journal, 343(Pt 1), 177–183.
Anbu, P., Gopinath, S. C. B., Cihan, A. C., & Chaulagain, B. P. (2013). Microbial enzymes and their applications in industries and medicine. BioMed Research International, 2013, 2–4.
Jaeger, K. E., & Reetz, M. T. (1998). Microbial lipases form versatile tools for biotechnology. Trends in Biotechnology, 16(9), 396–403.
Jaeger, K. E., Schneidinger, B., Rosenau, F., Werner, M., Lang, D., Dijkstra, B. W., et al. (1997). Bacterial lipases for biotechnological applications. Journal of Molecular Catalysis B Enzymatic, 3(1–4), 3–12.
Ray, A. (2012). Application of lipase in industry. Asian Journal of Pharmaceutical Technology, 2(2), 33–37.
Kim, J., Deng, L., Hong, E., & Ryu, Y. (2015). Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197. Protein Expression and Purification, 116, 90–97.
Kawamoto, T., Sonomoto, K., & Tanaka, A. (1987). Esterification in organic solvents: selection of hydrolases and effects of reaction conditions. Biocatalysis and Biotransformation, 1(2), 137–145.
Panda, T., & Gowrishankar, B. S. (2005). Production and applications of esterases. Applied Microbiology and Biotechnology, 67(2), 160–169.
Cowan, D. (1992). Enzymes from thermophilic archaebacteria: current and future applications in biotechnology. Biochemical Society Symposia, 58, 149–169.
Fiala, G., & Stetter, K. O. (1986). Pyrococcus furiosus; sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 °C. Archives of Microbiology, 145(1), 56–61.
Ikeda, M., & Clark, D. S. (1998). Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli. Biotechnology and Bioengineering, 57(5), 624–629.
Beaucage, S. L., & Iyer, R. P. (1992). Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron, 48(12), 2223–2311.
Damásio, A. R. L., Braga, C. M. P., Brenelli, L. B., Citadini, A. P., Mandelli, F., Cota, J., et al. (2013). Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus. Applied Microbiology and Biotechnology, 97(15), 6759–6767.
Shih, T.-W., & Pan, T.-M. (2011). Substitution of Asp189 residue alters the activity and thermostability of Geobacillus sp. NTU 03 lipase. Biotechnology Letters, 33(9), 1841–1846.
Sande, D., Souza, L. T. A., Oliveira, J. S., Santoro, M. M., Lacerda, I. C. A., Colen, G., et al. (2015). Colletotrichum gloeosporioides lipase: characterization and use in hydrolysis and esterifications. African Journal of Microbiology Research, 9(19), 1322–1330.
Mandelli, F., Franco Cairo, J. P. L., Citadini, A. P. S., Büchli, F., Alvarez, T. M., Oliveira, R. J., et al. (2013). The characterization of a thermostable and cambialistic superoxide dismutase from thermus filiformis. Letters in Applied Microbiology, 57(1), 40–46.
Hammersley, A. P. (1997). FIT2D: an introduction and overview. ESRF Internal Report ESRF97HA02.
Alvarez, T. M., Goldbeck, R., dos Santos, C. R., Paixão, D. A. A., Gonçalves, T. A., Franco Cairo, J. P. L., et al. (2013). Development and biotechnological application of a novel endoxylanase family GH10 identified from sugarcane soil metagenome. PLoS One, 8(7), e70014.
Svergun, D. I. (1992). Determination of the regularization parameter in indirect-transform methods using perceptual criteria. Journal of Applied Crystallography, 25(pt 4), 495–503.
Fischer, H., de Oliveira Neto, M., Napolitano, H. B., Polikarpov, I., & Craievich, A. F. (2009). Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale. Journal of Applied Crystallography, 43(1), 101–109.
Kozin, M. B., & Svergun, D. I. (2001). Automated matching of high- and low-resolution structural models. Journal of Applied Crystallography, 34(1), 33–41.
Svergun, D., Barberato, C., & Koch, M. H. (1995). CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. Journal of Applied Crystallography, 28(6), 768–773.
Rabbani, M., Bagherinejad, M. R., Sadeghi, H. M., Shariat, Z. S., Etemadifar, Z., Moazen, F., et al. (2013). Isolation and characterization of novel thermophilic lipase-secreting bacteria. Brazilian Journal of Microbiology, 44(4), 1113–1119.
Wang, L., Mavisakalyan, V., Tillier, E. R. M., Clark, G. W., Savchenko, A. V., Yakunin, A. F., et al. (2010). Mining bacterial genomes for novel arylesterase activity. Microbial Biotechnology, 3(6), 677–690.
Contesini, F. J., Calzado, F., Madeira, J. V., Rubio, M. V., Zubieta, M. P., de Melo, R. R., et al. (2016). Aspergillus lipases: Biotechnological and industrial application. In J. M. Mérillon & K. G. Ramawat (Eds.), Reference series in phytochemistry: Fungal metabolites (pp. 1–28). Cham, Switzerland: Springer International Publishing.
Bornscheuer, U. T. (2002). Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiology Reviews, 26(1), 73–81.
Fullbrook, P. (1996). Practical applied kinetics, industrial enzymology (2nd ed.). New York: Stockholm Press.
Kelly, S., & Price, N. (2000). The use of circular dichroism in the investigation of protein structure and function. Current Protein and Peptide Science, 1(4), 349–384.
Greenfield, N. J. (2006). Using circular dichroism spectra to estimate protein secondary structure. Nature Protocols, 1(6), 2876–2890.
Guinier, A., & Fournet, G. (1955). Small angle scattering of X-rays. Journal of Polymer Science, 1, 268.
Schubot, F. D., Kataeva, I. A., Blum, D. L., Shah, A. K., Ljungdahl, L. G., Rose, J. P., et al. (2001). Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum. Biochemistry, 40(42), 12524–12532.
Carvalho, C. M. L., & Cabral, J. M. S. (2000). Reverse micelles as reaction media for lipases. Biochimie, 82, 1063–1085.
Chen, Q., Luan, Z.-J., Cheng, X., & Xu, J.-H. (2015). Molecular dynamics investigation of the substrate binding mechanism in carboxylesterase. Biochemistry, 54(9), 1841–1848.
Rattray, F. P., & Fox, P. F. (1997). Purification and characterisation of an intracellular aminopeptidase from Brevibacterium linens ATCC 9174. Lait.
Fuciños, P., Atanes, E., López-López, O., Esperanza Cerdán, M., Isabel González-Siso, M., Pastrana, L., et al. (2011). Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability. Protein Expression and Purification, 78(2), 120–130.
Ayna, Ç., Kolcuoğlu, Y., Öz, F., Colak, A., & Ertunga, N. S. (2013). Purification and characterization of a pH and heat stable esterase from Geobacillus sp. TF17. TurkJBiochem, 4685, 329–336.
Almeida, R., Alqueres, S., Larentis, A., Rossle, S., Cardoso, A., Almeida, W., et al. (2006). Cloning, expression, partial characterization and structural modeling of a novel esterase from Pyrococcus furiosus. Enzyme and Microbial Technology, 39(5), 1128–1136.
Chandrayan, S. K., Dhaunta, N., & Guptasarma, P. (2008). Expression, purification, refolding and characterization of a putative lysophospholipase from Pyrococcus furiosus: retention of structure and lipase/esterase activity in the presence of water-miscible organic solvents at high temperatures. Protein Expression and Purification, 59(2), 327–333.
Nam, J.-K., Park, Y.-J., & Lee, H.-B. (2013). Cloning, expression, purification, and characterization of a thermostable esterase from the archaeon Sulfolobus solfataricus P1. Journal of Molecular Catalysis B: Enzymatic, 94, 95–103.
Torres, S., Baigorí, M. D., Swathy, S. L., Pandey, A., & Castro, G. R. (2009). Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase. Food Research International, 42(4), 454–460.
Yu, S., Zheng, B., Zhao, X., & Feng, Y. (2010). Gene cloning and characterization of a novel thermophilic esterase from Fervidobacterium nodosum Rt17-B1. Acta Biochimica et Biophysica Sinica, 42(4), 288–295.
Levisson, M., van der Oost, J., & Kengen, S. W. M. (2007). Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima. The FEBS Journal, 274(11), 2832–2842.
Córdova, J., Ryan, J. D., Boonyaratanakornkit, B. B., & Clark, D. S. (2008). Esterase activity of bovine serum albumin up to 160 °C: a new benchmark for biocatalysis. Enzyme and Microbial Technology, 42(3), 278–283.
Tamalampudi, S., Hama, S., Tanino, T., Talukder, M. R., Kondo, A., & Fukuda, H. (2007). Immobilized recombinant Aspergillus oryzae expressing heterologous lipase: an efficient whole-cell biocatalyst for enantioselective transesterification in non-aqueous medium. Journal of Molecular Catalysis B: Enzymatic, 48(1–2), 33–37.
Mustranta, A., Forssell, P., & Poutanen, K. (1993). Applications of immobilized lipases to transesterification and esterification reactions in nonaqueous systems. Enzyme and Microbial Technology, 15(2), 133–139.
Marchot, P., & Chatonnet, A. (2012). Enzymatic activity and protein interactions in alpha/beta hydrolase fold proteins: moonlighting versus promiscuity. Protein and Peptide Letters, 19(2), 132–143.
Guo, J., Zheng, X., Xu, L., Liu, Z., Xu, K., Li, S., et al. (2010). Characterization of a novel esterase Rv0045c from Mycobacterium tuberculosis. PLoS One, 5(10), e13143.
Rhee, J. K., Kim, D. Y., Ahn, D. G., Yun, J. H., Jang, S. H., Shin, H. C., et al. (2006). Analysis of the thermostability determinants of hyperthermophilic esterase EstE1 based on its predicted three-dimensional structure. Applied and Environmental Microbiology, 72(4), 3021–3025.
Dang, G., Chen, L., Li, Z., Deng, X., Cui, Y., Cao, J., et al. (2015). Expression, purification and characterisation of secreted esterase Rv2525c from Mycobacterium tuberculosis. Applied Biochemistry and Biotechnology, 176(1), 1–12.
Benavente, R., Esteban-Torres, M., Acebrón, I., De Las Rivas, B., Muñoz, R., Álvarez, Y., et al. (2013). Structure, biochemical characterization and analysis of the pleomorphism of carboxylesterase Cest-2923 from Lactobacillus plantarum WCFS1. FEBS Journal, 280(24), 6658–6671.
Li, P. Y., Ji, P., Li, C. Y., Zhang, Y., Wang, G. L., Zhang, X. Y., et al. (2014). Structural basis for dimerization and catalysis of a novel sterase from the GTSAG motif subfamily of the bacterial hormone-sensitive lipase family. Journal of Biological Chemistry, 289(27), 19031–19041.
Gupta, M. N. (1992). Enzyme function in organic solvents. European Journal of Biochemistry/FEBS, 203(1992), 25–32.
Dharmsthiti, S., & Luchai, S. (1999). Production, purification and characterization of thermophilic lipase from Bacillus sp. THL027. FEMS Microbiology Letters, 179(2), 241–246.
Pastore, G. M., Costa, V. D., & Koblitz, M. G. (2003). Production, partial purification and biochemical characterization of a novell Rhizopus sp. strain lipase. Food Science and Technology (Campinas), 23(2), 135–140.
Acknowledgments
The authors would like to thank the Brazilian National Council for Scientific Research (CNPq) for financial support (310186/2014-5, 442333/2014-5, 313749/2014-0, and 140796/2013-4).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Mandelli, F., Gonçalves, T.A., Gandin, C.A. et al. Characterization and Low-Resolution Structure of an Extremely Thermostable Esterase of Potential Biotechnological Interest from Pyrococcus furiosus . Mol Biotechnol 58, 757–766 (2016). https://doi.org/10.1007/s12033-016-9975-5
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-016-9975-5