Purification and identification of cutinases from <Emphasis Type="Italic">Colletotrichum kahawae</Emphasis> and <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> |
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Authors: | Zhenjia Chen Catarina F Franco Ricardo P Baptista Joaquim M S Cabral Ana V Coelho Jr" target="_blank">Carlos J RodriguesJr Eduardo P Melo |
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Institution: | 1.Centro de Investiga??o das Ferrugens do Cafeeiro, IICT,Oeiras,Portugal;2.Environment and Plant Protection Institute,Chinese Academy of Tropical Agriculture Sciences,Hainan,People’s Republic of China;3.Instituto Tecnologia Química e Biológica,Universidade Nova Lisboa,Oeiras,Portugal;4.Centro de Engenharia Biológica e Química,Instituto Superior Tecnico,Lisbon,Portugal;5.Universidade de évora,évora,Portugal;6.Centro Biomedicina Molecular e Estrutural,Universidade de Algarve,Faro,Portugal |
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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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Keywords: | Colletotrichum kahawae Colletotrichum gloeosporioides Carboxylesterase Cutinase Protein purification Mass spectrometry |
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