title: Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides.
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.
authors.affiliation: Centro de Investigação das Ferrugens do Cafeeiro, IICT, Quinta do Marquês, 2784-505, Oeiras, Portugal.
authors.forename: Zhenjia
authors.initials: Z
authors.lastname: Chen
authors.forename: Catarina F
authors.initials: CF
authors.lastname: Franco
authors.forename: Ricardo P
authors.initials: RP
authors.lastname: Baptista
authors.forename: Joaquim M S
authors.initials: JM
authors.lastname: Cabral
authors.forename: Ana V
authors.initials: AV
authors.lastname: Coelho
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Rodrigues
authors.forename: Eduardo P
authors.initials: EP
authors.lastname: Melo
mesh_terms: D000595:Amino Acid Sequence; D043182:Carboxylesterase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002853:Chromatography, Liquid; D020231:Colletotrichum / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D043182:Carboxylesterase