A novel strategy for the production of fluorescent enterocin CRL35 derivates

MASIAS E; ACUÑA L; MORERO R; MINAHK C

San Javier

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

Bacteriocins are antimicrobial peptides produced by prokaryotes, being of particular interest those synthesized by lactic bacteria, as many of them are considered GRAS (Generally Recognized As Safe). Enterocin CRL35 is a bacteriocin produced by Enterococcus mundtii CRL35, a strain isolated from an Argentinean artisanal cheese (Tafi del Valle, Tucumán). This antimicrobial peptide was tested as biopreservative agent in cheese, proving to be effective in controlling the growth of the food-borne pathogen Listeria monocytogenes. Furthermore, enterocin CRL35 displayed a synergistic effect with conventional antibiotics like erythromycin, chloramphenicol and tetracycline, a potential clinical application was suggested. The aim of the present work to establish a strategy for obtaining fluorescent derivatives of recombinant enterocin CRL35 in order to perform structurefunction studies, binding studies among others. As a first step, munA (enterocin CRL35 structural gene) was cloned into pET22b(+) vector. The bacteriocin was overexpressed in Escherichia coli Rosetta pLysS (DE3)grown in minimal medium and peptide was recovered from culture supernatants. Enterocin was purified by ammonium sulfate precipitation followed by reverse phase chromatography (C18-HPLC). Then, recombinant enterocin CRL35 was analyzed in detailed by mass spectrometry (MALDI-TOF and nanoLC-ESI MS/MS). It was confirmed that this peptide was correctly synthesized even with the intamolecular disulfide bond. Finally, enterocin CRL35 was derivatized with fluorescamine. This probe only fluoresces upon reaction with NH2 groups. As a consequence, fluorescent enterocin was identified by following emission fluorescence of the probe. This strategy will allow a fast and reliable way for producing derivatized bacteriocins, which will be ready for use in biophysical and biochemical studies.