Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides

• Published in: Applied microbiology and biotechnology Vol. 73; no. 6; pp. 1306 - 1313
• Main Authors: Chen, Zhenjia, Franco, Catarina F, Baptista, Ricardo P, Cabral, Joaquim M. S, Coelho, Ana V, Rodrigues, Carlos J. Jr, Melo, Eduardo P
• Format: Journal Article
• Language: English
• Published: Berlin Berlin/Heidelberg : Springer-Verlag 2007; Springer; Springer Nature B.V
• Subjects: Acetic acid; Amino Acid Sequence; Ammonium; Bacteria; Biological and medical sciences; Biotechnology; Carbon sources; carboxylesterase; Carboxylesterase - chemistry; Carboxylesterase - isolation & purification; Carboxylesterase - metabolism; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - isolation & purification; Carboxylic Ester Hydrolases - metabolism; Chromatography, Liquid; Coffee; Colletotrichum - enzymology; Colletotrichum gloeosporioides; Colletotrichum kahawae; cutinase; Electrophoresis, Polyacrylamide Gel; Fermentation; Fundamental and applied biological sciences. Psychology; Glomerella cingulata; Ions; Mass spectrometry; Molecular Sequence Data; Molecular Weight; Peptides; Protein purification; Proteins; Scientific imaging; Sequence Analysis, Protein; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Fungi; Identification; Purification; Fungi Imperfecti; Colletotrichum gloeosporioides; Thallophyta
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-006-0605-1

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.