Skip to main content
SpringerLink
Log in

Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The gene encoding carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima (tm0053) was cloned. The recombinant protein (EST53) was overexpressed in Escherichia coli without its NH2-terminal hydrophobic region, and with a C-terminal hexahistidine sequence. The enzyme was purified to homogeneity by heat treatment, followed by Ni2+ affinity chromatography, and then characterized. Among the p-nitrophenyl esters tested, the best substrate was p-nitrophenyl decanoate with K m and k cat values of 3.1 μM and 10.8 s−1, respectively, at 60°C and pH 7.5. The addition of O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid decreased the esterase activity, indicating that EST53 is dependent on the presence of Ca2+ ion. In addition, its activity was inhibited by the addition of phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, indicating that it contains serine and histidine residues, which play key roles in the catalytic mechanism. EST53 shows a relatively high degree of similarity to Burkholderia lipases that belong to family I.2 of the lipolytic enzymes, whereas the local sequence around the pentapeptide of EST53 is most similar to those of Bacillus lipases belonging to family I.4.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

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

    Article  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  Google Scholar 

  • Franken SM, Rozeboom HJ, Kalk KH, Dijkstra BW (1991) Crystal structure of haloalkane dehalogenase: an enzyme to detoxify halogenated alkanes. EMBO J 10:1297–1302

    Article  CAS  Google Scholar 

  • Frenken LG, Bos JW, Visser C, Muller W, Tommassen J, Verrips CT (1993a) An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase. Mol Microbiol 9:579–589

    Article  CAS  Google Scholar 

  • Frenken LG, de Groot A, Tommassen J, Verrips CT (1993b) Role of the lipB gene product in the folding of the secreted lipase of Pseudomonas glumae. Mol Microbiol 9:591–599

    Article  CAS  Google Scholar 

  • Fushinobu S, Jun SY, Hidaka M, Nojiri H, Yamane H, Shoun H, Omori T, Wakagi T (2005) A series of crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with various cleavage products. Biosci Biotechnol Biochem 69:491–498

    Article  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  CAS  Google Scholar 

  • Jaeger KE, Reetz MT (1998) Microbial lipases form versatile tools for biotechnology. Trends Biotechnol 16:396–403

    Article  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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Lang D, Hofmann B, Haalck L, Hecht HJ, Spener F, Schmid RD, Schomburg D (1996) Crystal structure of a bacterial lipase from Chromobacterium viscosum ATCC 6918 refined at 1.6 angstroms resolution. J Mol Biol 259:704–717

    Article  CAS  Google Scholar 

  • Liao DI, Remington SJ (1990) Structure of wheat serine carboxypeptidase II at 3.5-Å resolution. A new class of serine proteinase. J Biol Chem 265:6528–6531

    CAS  PubMed  Google Scholar 

  • Manco G, Giosue E, D’Auria S, Herman P, Carrea G, Rossi M (2000) Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus. Arch Biochem Biophys 373:182–192

    Article  CAS  Google Scholar 

  • Mandrich L, Merone L, Pezzullo M, Cipolla L, Nicotra F, Rossi M, Manco G (2005) Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family. J Mol Biol 345:501–512

    Article  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  CAS  Google Scholar 

  • Nardini M, Lang DA, Liebeton K, Jaeger KE, Dijkstra BW (2000) Crystal structure of Pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases. J Biol Chem 275:31219–31225

    Article  CAS  Google Scholar 

  • Noble ME, Cleasby A, Johnson LN, Egmond MR, Frenken LG (1993) The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveals a partially redundant catalytic aspartate. FEBS Lett 331:123–128

    Article  CAS  Google Scholar 

  • Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M, Remington SJ, Silman I, Schrag J et al (1992) The alpha/beta hydrolase fold. Protein Eng 5:197–211

    Article  CAS  Google Scholar 

  • Park OJ, Lee SH (2005) Stereoselective lipases from Burkholderia sp., cloning and their application to preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for (R)-Metalaxyl. J Biotechnol 120:174–182

    Article  CAS  Google Scholar 

  • Pathak D, Ollis D (1990) Refined structure of dienelactone hydrolase at 1.8 Å. J Mol Biol 214:497–525

    Article  CAS  Google Scholar 

  • Rashid N, Shimada Y, Ezaki S, Atomi H, Imanaka T (2001) Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A. Appl Environ Microbiol 67:4064–4069

    Article  CAS  Google Scholar 

  • Schrag JD, Li Y, Cygler M, Lang D, Burgdorf T, Hecht HJ, Schmid R, Schomburg D, Rydel TJ, Oliver JD, Strickland LC, Dunaway CM, Larson SB, Day J, McPherson A (1997) The open conformation of a Pseudomonas lipase. Structure 5:187–202

    Article  CAS  Google Scholar 

  • Simons JW, van Kampen MD, Ubarretxena-Belandia I, Cox RC, Alves dos Santos CM, Egmond MR, Verheij HM (1999) Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants. Biochemistry (Mosc) 38:2–10

    Article  CAS  Google Scholar 

  • Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I (1991) Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science 253:872–879

    Article  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  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Satoshi Kakugawa

    Present address: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

Authors and Affiliations

  1. Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Satoshi Kakugawa, Shinya Fushinobu, Takayoshi Wakagi & Hirofumi Shoun

Authors
  1. Satoshi Kakugawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shinya Fushinobu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takayoshi Wakagi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hirofumi Shoun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hirofumi Shoun.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kakugawa, S., Fushinobu, S., Wakagi, T. et al. Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima . Appl Microbiol Biotechnol 74, 585–591 (2007). https://doi.org/10.1007/s00253-006-0687-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-006-0687-9

Keywords

Search

Navigation