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Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus

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Abstract

Two genes encoding esterases EstA and EstB of Picrophilus torridus were identified by the means of genome analysis and were subsequently cloned in Escherichia coli. PTO 0988, which is encoding EstA, consists of 579 bp, whereas PTO 1141, encoding EstB, is composed of 696 bp, corresponding to 192 aa and 231 aa, respectively. Sequence comparison revealed that both biocatalysts have low sequence identities (14 and 16%) compared to previously characterized enzymes. Detailed analysis suggests that EstA and EstB are the first esterases from thermoacidophiles not classified as members of the HSL family. Furthermore, the subunits with an apparent molecular mass of 22 and 27 kDa of the homotrimeric EstA and EstB, respectively, represent the smallest esterase subunits from thermophilic microorganisms reported to date. The recombinant esterases were purified by Ni2+ affinity chromatography, and the activity of the purified esterases was measured over a wide pH (pH 4.5–8.5) and temperature range (10–90°C). Highest activity of the esterases was measured at 70°C (EstA) and 55°C (EstB) with short pNP-esters as preferred substrates. In addition, esters of the non-steroidal anti-inflammatory drugs naproxen, ketoprofen, and ibuprofen are hydrolyzed by both EstA and EstB. Extreme thermostability was measured for both enzymes at temperatures as high as 90°C. The determined half-life (t 1/2) at 90°C was 21 and 10 h for EstA and EstB, respectively. Remarkable preservation of esterase activity in the presence of detergents, urea, and commonly used organic solvents complete the exceptional phenotype of EstA and EstB.

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References

  • Altschul SF, Lipman DJ (1990) Protein database searches for multiple alignments. Proc Natl Acad Sci USA 87:5509–5513

    Article  PubMed  CAS  Google Scholar 

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343(Pt 1):177–183

    Article  PubMed  CAS  Google Scholar 

  • Bates PA, Kelley LA, MacCallum RM, Sternberg MJ (2001) Enhancement of protein modeling by human intervention in applying the automatic programs 3D-JIGSAW and 3D-PSSM. Proteins Suppl 5:39–46

    Article  CAS  Google Scholar 

  • Bell PJ, Sunna A, Gibbs MD, Curach NC, Nevalainen H, Bergquist PL (2002) Prospecting for novel lipase genes using PCR. Microbiology 148:2283–2291

    PubMed  CAS  Google Scholar 

  • Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

    Article  PubMed  CAS  Google Scholar 

  • Bernstein HJ (2000) Recent changes to RasMol, recombining the variants. Trends Biochem Sci 25:453–455

    Article  PubMed  CAS  Google Scholar 

  • Berriman M, Rutherford K (2003) Viewing and annotating sequence data with Artemis. Brief Bioinform 4:124–132

    Article  PubMed  CAS  Google Scholar 

  • Bornscheuer UT (2002) Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev 26:73–81

    Article  PubMed  CAS  Google Scholar 

  • Bourdineaud JP, Nehme B, Tesse S, Lonvaud-Funel A (2003) The ftsH gene of the wine bacterium Oenococcus oeni is involved in protection against environmental stress. Appl Environ Microbiol 69:2512–2520

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Britton HTS, Robinson RA (1931) Universal buffer solutions and the dissociation constant of veronal. J Chem Soc 458:1456–1462

    Article  Google Scholar 

  • Bull AT, Bunch AW, Robinson GK (1999) Biocatalysts for clean industrial products and processes. Curr Opin Microbiol 2:246–251

    Article  PubMed  CAS  Google Scholar 

  • Carvalho PdO, Contesini FJ, Bizaco R, Calafatti SA, Macedo GA (2006) Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger. J Ind Microbiol Biotechnol 33:713–718

    Article  CAS  Google Scholar 

  • Chen YS, Lee GC, Shaw JF (2006) Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli. J Agric Food Chem 54:7098–7104

    Article  PubMed  CAS  Google Scholar 

  • Ciaramella M, Napoli A, Rossi M (2005) Another extreme genome: how to live at pH 0. Trends Microbiol 13:49–51

    Article  PubMed  CAS  Google Scholar 

  • De Simone G, Galdiero S, Manco G, Lang D, Rossi M, Pedone C (2000) A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase. J Mol Biol 303:761–771

    Article  PubMed  CAS  Google Scholar 

  • Demirjian DC, Moris-Varas F, Cassidy CS (2001) Enzymes from extremophiles. Curr Opin Chem Biol 5:144–151

    Article  PubMed  CAS  Google Scholar 

  • Dharmsthiti S, Pratuangdejkul J, Theeragool GT, Luchai S (1998) Lipase activity and gene cloning of Acinetobacter calcoaceticus LP009. J Gen Appl Microbiol 44:139–145

    Article  PubMed  CAS  Google Scholar 

  • Di Giulio M (2005) Structuring of the genetic code took place at acidic pH. J Theor Biol 237:219–226

    Article  PubMed  CAS  Google Scholar 

  • Dutta S, Berman HM (2005) Large macromolecular complexes in the Protein Data Bank: a status report. Structure 13:381–388

    Article  PubMed  CAS  Google Scholar 

  • Ejima K, Liu J, Oshima Y, Hirooka K, Shimanuki S, Yokota Y, Hemmi H, Nakayama T, Nishino T (2004) Molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, Sulfolobus shibatae DSM5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme. J Biosci Bioeng 98:445–451

    PubMed  CAS  Google Scholar 

  • Finn RD, Mistry J, Schuster-Bockler B, Griffiths-Jones S, Hollich V, Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R et al (2006) Pfam: clans, web tools and services. Nucleic Acids Res 34:D247–D251

    Article  PubMed  CAS  Google Scholar 

  • Fischer M, Thai QK, Grieb M, Pleiss J (2006) DWARF—a data warehouse system for analyzing protein families. BMC Bioinformatics 7:495

    Article  PubMed  CAS  Google Scholar 

  • Futterer O, Angelov A, Liesegang H, Gottschalk G, Schleper C, Schepers B, Dock C, Antranikian G, Liebl W (2004) Genome sequence of Picrophilus torridus and its implications for life around pH 0. Proc Natl Acad Sci USA 101:9091–9096

    Article  PubMed  CAS  Google Scholar 

  • Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784–3788

    Article  PubMed  CAS  Google Scholar 

  • GOLD (2006) The Genomes On Line Database. Available at http://www.genomesonline.org. Accessed October 11 2006

  • Golyshina OV, Golyshin PN, Timmis KN, Ferrer M (2006) The ‘pH optimum anomaly’ of intracellular enzymes of Ferroplasma acidiphilum . Environ Microbiol 8:416–425

    Article  PubMed  CAS  Google Scholar 

  • Hotelier T, Renault L, Cousin X, Negre V, Marchot P, Chatonnet A (2004) ESTHER, the database of the alpha/beta-hydrolase fold superfamily of proteins. Nucleic Acids Res 32:D145–D147

    Article  PubMed  CAS  Google Scholar 

  • Hotta Y, Ezaki S, Atomi H, Imanaka T (2002) Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon. Appl Environ Microbiol 68:3925–3931

    Article  PubMed  CAS  Google Scholar 

  • Huddleston S, Yallop CA, Charalambous BM (1995) The identification and partial characterisation of a novel inducible extracellular thermostable esterase from the archaeon Sulfolobus shibatae. Biochem Biophys Res Commun 216:495–500

    Article  PubMed  CAS  Google Scholar 

  • Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O (1994) Bacterial lipases. FEMS Microbiol Rev 15:29–63

    Article  PubMed  CAS  Google Scholar 

  • Jaeger KE, Dijkstra BW, Reetz MT (1999) Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53:315–351

    Article  PubMed  CAS  Google Scholar 

  • Kim S, Lee SB (2004) Thermostable esterase from a thermoacidophilic archaeon: purification and characterization for enzymatic resolution of a chiral compound. Biosci Biotechnol Biochem 68:2289–2298

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Manco G, Di Gennaro S, De Rosa M, Rossi M (1994) Purification and characterization of a thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius. Eur J Biochem 221:965–972

    Article  PubMed  CAS  Google Scholar 

  • Manco G, Adinolfi E, Pisani FM, Ottolina G, Carrea G, Rossi M (1998) Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily. Biochem J 332(Pt 1):203–212

    PubMed  CAS  Google Scholar 

  • Manco G, Camardella L, Febbraio F, Adamo G, Carratore V, Rossi M (2000) Homology modeling and identification of serine 160 as nucleophile of the active site in a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus. Protein Eng 13:197–200

    Article  PubMed  CAS  Google Scholar 

  • Mandrich L, Pezzullo M, Rossi M, Manco G (2006) SSoNΔ and SSoNΔlong: two thermostable esterases from the same ORF in the archaeon Sulfolobus solfataricus? Archaea 2:1–7

    Article  Google Scholar 

  • Margolin AL (1993) Enzymes in the synthesis of chiral drugs. Enzyme Microb Technol 15:266–280

    Article  PubMed  CAS  Google Scholar 

  • Morana A, Di Prizito N, Aurilia V, Rossi M, Cannio R (2002) A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein. Gene 283:107–115

    Article  PubMed  CAS  Google Scholar 

  • Morel F, Delmas F, Jobin MP, Divies C, Guzzo J (2001) Improved acid tolerance of a recombinant strain of Escherichia coli expressing genes from the acidophilic bacterium Oenococcus oeni. Lett Appl Microbiol 33:126–130

    Article  PubMed  CAS  Google Scholar 

  • Park YJ, Choi SY, Lee HB (2006) A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression. Biochim Biophys Acta 1760:820–828

    PubMed  CAS  Google Scholar 

  • Rhee JK, Ahn DG, Kim YG, Oh JW (2005) New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library. Appl Environ Microbiol 71:817–825

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Sayle RA, Milner-White EJ (1995) RASMOL: biomolecular graphics for all. Trends Biochem Sci 20:374

    Article  PubMed  CAS  Google Scholar 

  • Schepers B, Thiemann V, Antranikian G (2006) Characterization of a novel glucoamylase from the thermoacidophilic Archaeon Picrophilus torridus heterologously expressed in E. coli. Eng Life Sci 6:311–317

    Article  CAS  Google Scholar 

  • Schmidt-Dannert C, Sztajer H, Stocklein W, Menge U, Schmid RD (1994) Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus. Biochim Biophys Acta 1214:43–53

    PubMed  CAS  Google Scholar 

  • Schomburg I, Chang A, Ebeling C, Gremse M, Heldt C, Huhn G, Schomburg D (2004) BRENDA, the enzyme database: updates and major new developments. Nucleic Acids Res 32:D431–D433

    Article  PubMed  CAS  Google Scholar 

  • Schutte M, Fetzner S (2007) EstA from Arthrobacter nitroguajacolicus Ru61a, a thermo- and solvent-tolerant carboxylesterase related to Class C beta-lactamases. Curr Microbiol

  • Serour E, Antranikian G (2002) Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum, Picrophilus torridus and Picrophilus oshimae . Antonie Van Leeuwenhoek 81:73–83

    Article  PubMed  CAS  Google Scholar 

  • Sobek H, Gorisch H (1988) Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochem J 250:453–458

    PubMed  CAS  Google Scholar 

  • Suzuki Y, Miyamoto K, Ohta H (2004) A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. FEMS Microbiol Lett 236:97–102

    Article  PubMed  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  PubMed  CAS  Google Scholar 

  • Vossenberg vd, Driessen AJ, Zillig W, Konings WN (1998) Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae. Extremophiles 2:67–74

    Article  Google Scholar 

  • Winkler UK, Stuckmann M (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol 138:663–670

    PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by grant 04-008 202131 from the German BMBF. We would like to thank Dr. Maryna Royter and Dr. Christian Elend for their support.

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Authors and Affiliations

  1. Genomics Division, DOE Joint Genome Institute, Walnut Creek, CA, USA

    Matthias Hess

  2. Institute of Technical Microbiology, Hamburg University of Technology, Kasernenstr. 12, 21073, Hamburg, Germany

    Moritz Katzer & Garabed Antranikian

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  1. Matthias Hess
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  2. Moritz Katzer
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Correspondence to Garabed Antranikian.

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Communicated by K. Horikoshi.

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Hess, M., Katzer, M. & Antranikian, G. Extremely thermostable esterases from the thermoacidophilic euryarchaeon Picrophilus torridus . Extremophiles 12, 351–364 (2008). https://doi.org/10.1007/s00792-008-0139-9

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