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
Article

Abstract

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.

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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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