Publication: Caracterización, diseño y producción recombinante de bacteriocinas y otros péptidos antimicrobianos, secuenciación genómica y efecto en la microbiota de péptidos bioactivos producidos por enterococos
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2017-05-23
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Universidad Complutense de Madrid
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Abstract
El trabajo desarrollado en esta tesis doctoral ha empleado técnicas de microbiología y bioquímica clásicas, así como técnicas recientes de ingeniería genética, genómica y proteómica y describe en capítulos diferenciados, los siguientes objetivos: En el capítulo III, se describe la identificación y caracterización de bacterias lácticas (BAL) aísladas de heces de buitres leonados (Gyps fulvus subesp. fulvus) por su actividad antimicrobiana, genes que codifican bacteriocinas, factores potenciales de virulencia, susceptibilidad a antibióticos, genotipado por las técnicas de ERIC-PCR y MLST y caracterización bioquímica de las bacteriocinas purificadas por MALDI-TOF MS, secuenciación aminoacídica N-terminal por degradación de Edman y secuenciación de novo de las bacteriocinas purificadas por MALDI TOF/TOF MS. De interés ha sido el aislamiento y caracterización de E. faecium M3K31, productor de la enterocina HF (EntHF) y de E. faecalis M3M42, que podría postularse como potencialmente productora de péptidos bioactivos. En el capítulo IV, se describe la clonación y expresión de genes sintéticos que codifican las bacteriocinas de amplio espectro de acción SRCAM 602, OR-7, E-760 y L-1077, previamente descritas por su actividad antimicrobiana frente a bacterias Gram-positivas y Gram-negativas incluyendo Campylobacter spp. y producidas por levaduras recombinantes, derivadas de P. pastoris. No obstante, las levaduras recombinantes no mostraron una actividad antimicrobiana directa y la potencialmente presente en los sobrenadantes de los cultivos productores, sólo pudo evaluarse mediante un procedimiento multicromatográfico de purificación. Sin embargo, el análisis de las fracciones purificadas por MALDI-TOF MS y MALDI TOF/TOF, reveló la presencia en las mismas de fragmentos peptídicos no relacionados con la masa molecular deducida de las bacteriocinas clonadas...
The research work carried out in this doctoral thesis has been based on the use of classical microbiological and biochemical techniques as well as recent genetic engineering techniques, genomics and proteomics and describes, in separated chapters, the following objectives: In chapter III, the identification and characterization of bacteriocinogenic lactic acid bacteria (LAB) isolated from the feces of Griffon vultures (Gyps fulvus subsp. fulvus), is described. All isolates were evaluated for their antimicrobial activity, genes coding for bacteriocins, potential virulence factors, antibiotic susceptibility and genotyping by ERIC-PCR and MLST. Purified bacteriocins were biochemically characterized by MALDI-TOF MS, N-terminal amino acid sequencing by Edman degradation, and de novo sequencing by MALDI TOF/TOF MS. It is worth to highlight the isolation and characterization of E. faecium M3K31, producer of enterocin HF (EntHF) and E. faecalis M3M42, that could be potentially proposed as producer of bioactive peptides. In chapter IV, synthetic genes encoding the broad antimicrobial spectrum bacteriocins SCRCAM 602, OR-7, E-760 and L-1077, previously described for their antimicrobial activity against Gram-positive and Gram-negative bacteria, including Campylobacter spp. were cloned in the protein expression vector pPICZA, for expression by P. pastoris. However, none of the recombinant yeasts showed a direct antimicrobial activity whereas the antimicrobial activity potentially present in the supernatants, could only be evaluated through the use of a multichromatographic purification procedure. Furthermore, analysis of the purified fractions by MALDI-TOF MS and MALFI TOF/TOF revealed the presence in the samples of peptide fragments, not related to the deduced molecular mass of the cloned bacteriocins...
The research work carried out in this doctoral thesis has been based on the use of classical microbiological and biochemical techniques as well as recent genetic engineering techniques, genomics and proteomics and describes, in separated chapters, the following objectives: In chapter III, the identification and characterization of bacteriocinogenic lactic acid bacteria (LAB) isolated from the feces of Griffon vultures (Gyps fulvus subsp. fulvus), is described. All isolates were evaluated for their antimicrobial activity, genes coding for bacteriocins, potential virulence factors, antibiotic susceptibility and genotyping by ERIC-PCR and MLST. Purified bacteriocins were biochemically characterized by MALDI-TOF MS, N-terminal amino acid sequencing by Edman degradation, and de novo sequencing by MALDI TOF/TOF MS. It is worth to highlight the isolation and characterization of E. faecium M3K31, producer of enterocin HF (EntHF) and E. faecalis M3M42, that could be potentially proposed as producer of bioactive peptides. In chapter IV, synthetic genes encoding the broad antimicrobial spectrum bacteriocins SCRCAM 602, OR-7, E-760 and L-1077, previously described for their antimicrobial activity against Gram-positive and Gram-negative bacteria, including Campylobacter spp. were cloned in the protein expression vector pPICZA, for expression by P. pastoris. However, none of the recombinant yeasts showed a direct antimicrobial activity whereas the antimicrobial activity potentially present in the supernatants, could only be evaluated through the use of a multichromatographic purification procedure. Furthermore, analysis of the purified fractions by MALDI-TOF MS and MALFI TOF/TOF revealed the presence in the samples of peptide fragments, not related to the deduced molecular mass of the cloned bacteriocins...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, leída el 14-10-2016