Applied Microbiology and Biotechnology

, Volume 73, Issue 6, pp 1306–1313 | Cite as

Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides

  • Zhenjia Chen
  • Catarina F. Franco
  • Ricardo P. Baptista
  • Joaquim M. S. Cabral
  • Ana V. Coelho
  • Carlos J. RodriguesJr.
  • Eduardo P. Melo
Biotechnologically Relevant Enzymes and Proteins

Abstract

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.

Keywords

Colletotrichum kahawae Colletotrichum gloeosporioides Carboxylesterase Cutinase Protein purification Mass spectrometry 

Notes

Acknowledgments

Zhenjia Chen acknowledges a fellowship from IICT (Instituto de Investigação Científica Tropical). Gonçalo Cabrita is acknowledged for English revision.

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Zhenjia Chen
    • 1
    • 2
    • 3
  • Catarina F. Franco
    • 3
  • Ricardo P. Baptista
    • 4
  • Joaquim M. S. Cabral
    • 4
  • Ana V. Coelho
    • 3
    • 5
  • Carlos J. RodriguesJr.
    • 1
  • Eduardo P. Melo
    • 4
    • 6
  1. 1.Centro de Investigação das Ferrugens do Cafeeiro, IICTOeirasPortugal
  2. 2.Environment and Plant Protection InstituteChinese Academy of Tropical Agriculture SciencesHainanPeople’s Republic of China
  3. 3.Instituto Tecnologia Química e BiológicaUniversidade Nova LisboaOeirasPortugal
  4. 4.Centro de Engenharia Biológica e QuímicaInstituto Superior TecnicoLisbonPortugal
  5. 5.Universidade de ÉvoraÉvoraPortugal
  6. 6.Centro Biomedicina Molecular e EstruturalUniversidade de AlgarveFaroPortugal

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