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

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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 (K m ) and V max 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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Acknowledgments

A.P. gratefully acknowledges support from ISAS for a PhD fellowship. We thank Corrado Guarnaccia and Sotir Zahariev for mass spectrometry analysis.

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Authors and Affiliations

  1. International School for Advanced Studies, Alessandro Pesaresi, Via Beirut 2-4, I-34014, Trieste, Italy

    Alessandro Pesaresi

  2. Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, I-34012, Trieste, Italy

    Giulia Devescovi, Vittorio Venturi & Giuliano Degrassi

  3. Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Sez. di Trieste, Area Science Park, Basovizza, S.S. 14 Km 163.5, I-34012, Trieste, Italy

    Doriano Lamba

Authors
  1. Alessandro Pesaresi
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  2. Giulia Devescovi
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  3. Doriano Lamba
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  4. Vittorio Venturi
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  5. Giuliano Degrassi
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Correspondence to Giuliano Degrassi.

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Pesaresi, A., Devescovi, G., Lamba, D. et al. Isolation, Characterization, and Heterologous Expression of a Carboxylesterase of Pseudomonas aeruginosa PAO1. Curr Microbiol 50, 102–109 (2005). https://doi.org/10.1007/s00284-004-4414-4

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  • DOI: https://doi.org/10.1007/s00284-004-4414-4

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