Examinando por Autor "Callegari, Eduardo"
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Ítem Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp(Elsevier Ltd, 2017) Oleas, Gabriela; Callegari, Eduardo; Sepúlveda, Romina; Eyzaguirre, JaimeThe lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10). © 2017 Elsevier LtdÍtem Identification of Clostridium difficile Immunoreactive Spore Proteins of the Epidemic Strain R20291(Wiley-VCH Verlag, 2018-09) Pizarro-Guajardo, Marjorie; Ravanal, María Cristina; Paez, Maria Daniela; Callegari, Eduardo; Paredes-Sabja, DanielPurpose: Clostridium difficile infections are the leading cause of diarrhea associated with the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. The development of vaccine and passive immunization therapies against C. difficile has focused on toxins A and B. In this study, an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C. difficile spores as potential candidates for the development of immunotherapy and/or diagnostic methods against this devastating infection is used. Experimental design: To identify potential immunogenic proteins on the surface of C. difficile R20291, spore coat/exosporium extracts are separated by 2D electrophoresis (2-DE) and analyzed for reactivity against C. difficile spore-specific goat sera. Finally, the selected spots are in-gel digested with chymotrypsin, peptides generated are separated by nanoUPLC followed by MS/MS using Quad-TOF-MS, corroborated by Ultimate 3000RS-nano-UHPLC coupled to Q-Exactive-Plus-Orbitrap MS. Results: The analysis identify five immunoreactive proteins: spore coat proteins CotE, CotA, and CotCB; exosporium protein CdeC; and a cytosolic methyltransferase. Conclusion: This data provides a list of spore surface protein candidates as antigens for vaccine development against C. difficile infections. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim