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Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides

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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.

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Acknowledgments

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

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Authors and Affiliations

  1. Centro de Investigação das Ferrugens do Cafeeiro, IICT, Quinta do Marquês, 2784-505, Oeiras, Portugal

    Zhenjia Chen & Carlos J. Rodrigues Jr.

  2. Environment and Plant Protection Institute, Chinese Academy of Tropical Agriculture Sciences, Hainan, People’s Republic of China

    Zhenjia Chen

  3. Instituto Tecnologia Química e Biológica, Universidade Nova Lisboa, Oeiras, Portugal

    Zhenjia Chen, Catarina F. Franco & Ana V. Coelho

  4. Centro de Engenharia Biológica e Química, Instituto Superior Tecnico, Av. Rovisco Pais, 1049-001, Lisbon, Portugal

    Ricardo P. Baptista, Joaquim M. S. Cabral & Eduardo P. Melo

  5. Universidade de Évora, Évora, Portugal

    Ana V. Coelho

  6. Centro Biomedicina Molecular e Estrutural, Universidade de Algarve, Campus de Gambelas, 8005-139, Faro, Portugal

    Eduardo P. Melo

Authors
  1. Zhenjia Chen
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  2. Catarina F. Franco
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  3. Ricardo P. Baptista
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  4. Joaquim M. S. Cabral
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  5. Ana V. Coelho
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  6. Carlos J. Rodrigues Jr.
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  7. Eduardo P. Melo
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Correspondence to Eduardo P. Melo.

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Chen, Z., Franco, C.F., Baptista, R.P. et al. Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides . Appl Microbiol Biotechnol 73, 1306–1313 (2007). https://doi.org/10.1007/s00253-006-0605-1

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  • DOI: https://doi.org/10.1007/s00253-006-0605-1

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