Current Microbiology

, Volume 50, Issue 2, pp 102–109

Isolation, Characterization, and Heterologous Expression of a Carboxylesterase of Pseudomonas aeruginosa PAO1

  • Alessandro Pesaresi
  • Giulia Devescovi
  • Doriano Lamba
  • Vittorio Venturi
  • Giuliano Degrassi


We purified to homogeneity an intracellular esterase from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The enzyme hydrolyzes p-nitrophenyl acetate and other acetylated substrates. The N-terminal amino acid sequence was analyzed and 11 residues, SEPLILDAPNA, were determined. The corresponding gene PA3859 was identified in the P. aeruginosa PAO1 genome as the only gene encoding for a protein with this N-terminus. The encoding gene was cloned in Escherichia coli, and the recombinant protein expressed and purified to homogeneity. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and analytical gel filtration chromatography, the esterase was found to be a monomer of approximately 24 kDa. The experimentally determined isoelectric point was 5.2 and the optimal enzyme activity was at 55°C and at pH 9.0. The esterase preferentially hydrolyzed short-chain fatty acids. It is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by ethylendiaminotetraacetic acid (EDTA). Native enzyme preparations typically showed a Michaelis constant (Km) and Vmax of 0.43 mM and 12,500 U mg−1, respectively, using p-nitrophenyl acetate as substrate. Homology-based database searches clearly revealed the presence of the consensus GXSXG signature motif that is present in the serine-dependent acylhydrolase protein family.

Literature Cited

  1. 1.
    Brady, L, Brzozowski, AM, Derewenda, ZS,  et al. 1990A serine protease triad forms the catalytic centre of a triacylglycerol lipaseNature343767770Google Scholar
  2. 2.
    Brenner, S 1988The molecular evolution of genes and proteins: A tale of two serinesNature334528530Google Scholar
  3. 3.
    Chao, YP, Fu, H, Wang, YL, Huang, WB, Wang, JY 2003Molecular cloning of the carboxylesterase gene and biochemical characterization of the encoded protein from Pseudomonas citronellolis ATCC 13674Res Microbiol154521526CrossRefGoogle Scholar
  4. 4.
    Degrassi, G, Uotila, L, Klima, R, Venturi, V 1999Purification and properties of an esterase from the yeast Saccharomyces cerevisiae and identification of the encoding geneAppl Environ Microbiol6534703472Google Scholar
  5. 5.
    Degrassi, G, Kojic, M, Ljubijankic, G, Venturi, V 2000The acetyl xylan esterase of Bacilluspumilus belongs to a family of esterases with broad substrate specificityMicrobiology14615851591PubMedGoogle Scholar
  6. 6.
    Devedjiev, Y, Dauter, Z, Kuznetsov, SR, Jones, TL, Derewenda, ZS 2000Crystal structure of the human acyl protein thioesterase I from a single X-ray data set to 1.5 AStruct Fold Des811371146CrossRefGoogle Scholar
  7. 7.
    Duncan, JA, Gilman, AG 1998A cytoplasmatic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21 (RAS)J BioI Chern2731583015837CrossRefGoogle Scholar
  8. 8.
    Harms, N, Ras, J, Reijnders, WNM, Spanning, RJM, Stouthhamer, AH 1996S-Formylglutathione hydrolase of Paracoccus denitrificans is homologous to human esterase D: A universal pathway for formaldehyde detoxificationJ Bacteriol17862966299Google Scholar
  9. 9.
    Hong, KH, Jang, WH, Choi, KD, Yoo, OJ 1991Characterization of Pseudomonas fluorescens carboxylesterase: Cloning and expression of the esterase gene in Escherichia coliAgr Biol Chem5528392845Google Scholar
  10. 10.
    Jakoby, WE, Ziegler, DM 1990The enzymes of detoxificationJ Biol Chem2652071520718Google Scholar
  11. 11.
    Johnson, LM, Talbot, HW 1983Detoxification of pesticides by microbial enzymesExperientia3912361246Google Scholar
  12. 12.
    Kim, YS, Lee, HE, Choi, KD, Park, S, Yoo, OJ 1994Cloning of Pseudomonasfluorescens carboxylesterase gene and characterization of its product expressed in Escherichia coliBiosci Biotech Biochem58111116Google Scholar
  13. 13.
    Kim, KK, Song, HK, Shin, DH, Hwang, KY, Choe, S, Yoo, OJ, Sub, SW 1997Crystal structure of carboxylesterase from Pseudomonas fluorescens, an alpha/beta hydrolase with broad substrate specificityStructure515711584CrossRefGoogle Scholar
  14. 14.
    Kim, HE, Lee, IS, Kim, JH, Hahn, KW, Park, VJ, Han, HS, Park, KR 2003Gene cloning, sequencing, and expression of an esterase from Acinetobacter lwoffii I6C-lCurr Microbiol46291295CrossRefGoogle Scholar
  15. 15.
    McRee, D 2003Protecting against cocaine, heroin, and sarin gasChem Biol10295297CrossRefGoogle Scholar
  16. 16.
    Laemmli, VK 1970Cleavage of structural protein during the assembly of the head of bacteriophage T4Nature227680685PubMedGoogle Scholar
  17. 17.
    Morana, A, Di Prizito, N, Aurilia, V, Rossi, M, Cannio, R 2002A carboxylesterase from the hyperthermophilic archeon Sulfolobus solfataricus: Cloning of the gene, characterization of the proteinGene283107115PubMedGoogle Scholar
  18. 18.
    Notredame, C, Higgins, D, Heringa, J 2000T-Coffee: A novel method for multiple sequence alignmentsJ Mol Biol302205217CrossRefPubMedGoogle Scholar
  19. 19.
    Petersen, EI, Valinger, G, Solkner, B, Stubenrauch, G, Schwab, H 2001A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to beta-lactamases and DD-peptidasesJ Biotechnol891125Google Scholar
  20. 20.
    Sambrook, J, Fritsch, EF, Maniatis, T 1989Molecular cloning: a laboratory manual2Cold Spring Harbor PressCold Spring Harbor, NYGoogle Scholar
  21. 21.
    Sanishvili, R, Yakunin, AF, Laskowski, RA, Skarina, T, Evdokimova, E, Doherty-Kirby, A, Lajoie, GA, Thornton, JM, Arrowsmith, CH, Savchenko, A, Joachimiak, A, Edwards, AM 2003Integrating structure, bioinformatics, and enzymology to discover function: BioH, a new carboxylesterase from Escherichia coliJ Biol Chem2782603226045CrossRefGoogle Scholar
  22. 22.
    Satoh, T, Hosokawa, M 1998The mammalian carboxylesterases: From molecules to functionsAnnu Rev Pharmacol Toxicol38257288CrossRefGoogle Scholar
  23. 23.
    Stover, CK, Pham, XQ, Erwin, AL, Mizoguchi, SD, Warrener, P, Hickey, MJ, Brinkman, FS, Hufnagle, WO, Kowalik, DJ, Lagrou, M, Garber, RL, Goltry, L, Tolentino, E, Westbrock-L Wadman, S, Yuan, Y, Brody, LL, Coulter, SN, Folger, KR, Kas, A, Larbig, K, Urn, R, Smith, K, Spencer, D, Wong, GK, Wu, Z, Paulsen, IT, Reizer, J, Saier, MH, Hancock, RE, Lory, S, Olson, MV 2000Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogenNature406959964CrossRefPubMedGoogle Scholar
  24. 24.
    Sugihara, A, Shimada, Y, Nagao, T, Iizumi, T, Nakamura, K, Fukase, T, Tominaga, Y 1994Purification and characterization of a carboxylesterase from Pseudomonas sp. KWI-56Biosci Biotechnol Biochem58752755Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Alessandro Pesaresi
    • 1
  • Giulia Devescovi
    • 2
  • Doriano Lamba
    • 3
  • Vittorio Venturi
    • 2
  • Giuliano Degrassi
    • 2
  1. 1.International School for Advanced StudiesAlessandro PesaresiTriesteItaly
  2. 2.Bacteriology GroupInternational Centre for Genetic Engineering and BiotechnologyTriesteItaly
  3. 3.Istituto di CristallografiaConsiglio Nazionale delle RicercheTriesteItaly

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