Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa
Digital
- Autores:
-
Pérez-Vergel, Astrid Carolina
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2024
- Institución:
- Universidad de Santander
- Repositorio:
- Repositorio Universidad de Santander
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.udes.edu.co:001/11343
- Palabra clave:
- Quorum sensing
Lactonasas
Pseudomonas aeruginosa
Autoinductores
Quorum Sensing
Lactonases
Pseudomonas aeruginosa
Autoinducers
- Rights
- embargoedAccess
- License
- http://purl.org/coar/access_right/c_f1cf
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| dc.title.spa.fl_str_mv |
Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa |
| dc.title.translated.none.fl_str_mv |
Characterization of the Epp 9 variant lactonase derived from the aiiA gene of Bacillus thuringiensis in the inhibition of the C4-HSL and 3-oxo-C12-HSL autoinducers of Pseudomonas aeruginosa. |
| title |
Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa |
| spellingShingle |
Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa Quorum sensing Lactonasas Pseudomonas aeruginosa Autoinductores Quorum Sensing Lactonases Pseudomonas aeruginosa Autoinducers |
| title_short |
Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa |
| title_full |
Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa |
| title_fullStr |
Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa |
| title_full_unstemmed |
Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa |
| title_sort |
Caracterización de la lactonasa variante Epp 9 derivada del gen aiiA de Bacillus thuringiensis en la inhibición de los autoinductores C4-HSL y 3-oxo-C12-HSL de Pseudomonas aeruginosa |
| dc.creator.fl_str_mv |
Pérez-Vergel, Astrid Carolina |
| dc.contributor.advisor.none.fl_str_mv |
Suárez-Barera, Miguel Orlando |
| dc.contributor.author.none.fl_str_mv |
Pérez-Vergel, Astrid Carolina |
| dc.contributor.jury.none.fl_str_mv |
Valdivieso-Quintero, Wilfredo Hernández-Peñaranda, Indira Paola |
| dc.contributor.researchgroup.none.fl_str_mv |
Laboratorio de Biología Molecular y Biotecnología |
| dc.subject.proposal.spa.fl_str_mv |
Quorum sensing Lactonasas Pseudomonas aeruginosa Autoinductores |
| topic |
Quorum sensing Lactonasas Pseudomonas aeruginosa Autoinductores Quorum Sensing Lactonases Pseudomonas aeruginosa Autoinducers |
| dc.subject.proposal.eng.fl_str_mv |
Quorum Sensing Lactonases Pseudomonas aeruginosa Autoinducers |
| description |
Digital |
| publishDate |
2024 |
| dc.date.issued.none.fl_str_mv |
2024-11-28 |
| dc.date.accessioned.none.fl_str_mv |
2025-01-31T21:04:23Z |
| dc.date.available.none.fl_str_mv |
2025-12-10 2025-01-31T21:04:23Z |
| dc.type.none.fl_str_mv |
Trabajo de grado - Pregrado |
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http://purl.org/coar/resource_type/c_7a1f |
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http://purl.org/coar/version/c_71e4c1898caa6e32 |
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Text |
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info:eu-repo/semantics/bachelorThesis |
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http://purl.org/redcol/resource_type/TP |
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Universidad de Santander |
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T 33.24 P272c |
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Repositorio Digital Universidad de Santander |
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https://repositorio.udes.edu.co |
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https://repositorio.udes.edu.co/handle/001/11343 |
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Universidad de Santander T 33.24 P272c Repositorio Digital Universidad de Santander |
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https://repositorio.udes.edu.co https://repositorio.udes.edu.co/handle/001/11343 |
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Biochemistry, 20(9), 2444-2449. Espinoza Pesantez, D. I., & Esparza Sanchez, G. F. (2021). Resistencia enzimática en Pseudomonas aeruginosa, aspectos clínicos y de laboratorio. Revista chilena de infectología, 38(1), 69-80. Fan X., Liu X. y Liu Y. (2012) Clonación y caracterización de una nueva esterasa termoestable derivada del metagenoma que actúa sobre lactonas de N -acilhomoserina . J. Mol. Catal. B. Enzym. 83 , 29–37 10.1016/j.molcatb.2012.07.006 Fetzner, S. (2015). Quorum quenching enzymes. Journal of biotechnology, 201, 2-14. Fuqua, W. C., Winans, S. C., & Greenberg, E. P. (1994). Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. Journal of bacteriology, 176(2), 269-275. Gallardo Castro, J. A. (2018). Determinación de la inhibición de quorum sensing por la variante lactonasa 852 sobre la producción de biopelículas en bacterias gram negativas multidrogoresistentes. García Gómez, D., & Aguiar Díaz, E. (2022). Caracterización de Pseudomonas aeruginosa en la Sala de Angiología. Acta Médica del Centro, 16(1), 46-57. García-Vargas, S. (2023). Diseño de variantes del gen aiiA de Bacillus thuringiensis mediante una técnica de evolución dirigida al sitio. Universidad de Santander. Ghanei-Motlagh, R., Mohammadian, T., Gharibi, D., Menanteau-Ledouble, S., Mahmoudi, E., Khosravi, M., Zarea, M., & El-Matbouli, M. (2019). Quorum Quenching Properties and Probiotic Potentials of Intestinal Associated Bacteria in Asian Sea Bass Lates calcarifer. Marine drugs, 18(1), 23. https://doi.org/10.3390/md18010023 Givskov M, de Nys R, Manefield M, Gram L, Maximilien R, Eberl L, Molin S, Steinberg PD, and Kjelleberg S (1996). Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling. Journal of Bacteriology 178, 6618–6622 Gomaa, S. E., Shaker, G. H., Mosallam, F. M., & Abbas, H. A. (2022). Knocking down Pseudomonas aeruginosa virulence by oral hypoglycemic metformin nano emulsion. World journal of microbiology & biotechnology, 38(7), 119. https://doi.org/10.1007/s11274-022-03302-8 Grandclément, C., Tannières, M., Moréra, S., Dessaux, Y., & Faure, D. (2016). Quorum quenching: role in nature and applied developments. FEMS microbiology reviews, 40(1), 86-116. Gurevich, D., Dor, S., Erov, M., Dan, Y., Moy, J. C., Mairesse, O., ... & Afriat-Jurnou, L. (2021). Directed enzyme evolution and encapsulation in peptide nanospheres of quorum quenching lactonase as an antibacterial treatment against plant pathogen. ACS Applied Materials & Interfaces, 13(2), 2179-2188. https://pubs.acs.org/doi/abs/10.1021/acsami.0c15808 Haramati, R., Dor, S., Gurevich, D., Levy, D., Freund, D., Rytwo, G., Sharon, I., & Afriat-Jurnou, L. (2022). Mining Marine Metagenomes Revealed a Quorum-Quenching Lactonase with Improved Biochemical Properties That Inhibits the Food Spoilage Bacterium Pseudomonas fluorescens. Applied and environmental microbiology, 88(4), e0168021. https://doi.org/10.1128/AEM.01680-21 Jiménez Tuta, D. A. (2020). Búsqueda de inhibidores de quorum sensing a partir de compuestos volátiles provenientes de aceites esenciales. Joseph W. St. Geme, Katherine A. Rempe. (2018). 172 - Haemophilus influenzae. Principles and Practice of Pediatric Infectious Diseases (Fifth Edition) Elsevier. Pages 926-931.e3 Khalid, S. J., Ain, Q., Khan, S. J., Jalil, A., Siddiqui, M. F., Ahmad, T., ... & Adnan, F. (2022). Targeting Acyl Homoserine Lactones (AHLs) by the quorum quenching bacterial strains to control biofilm formation in Pseudomonas aeruginosa. Saudi Journal of Biological Sciences, 29(3), 1673-1682. Kumar, N., & Chavan, S. K. (2023). Pseudomonas aeruginosa: un patógeno persistente y enfoques actuales de tratamiento microbiológico. Salud, Ciencia y Tecnología, 3, 404-404. Laborda, P., Sanz-García, F., Ochoa-Sánchez, L. E., Gil-Gil, T., Hernando-Amado, S., & Martínez, J. L. (2022). Wildlife and Antibiotic Resistance. Frontiers in cellular and infection microbiology, 12, 873989. https://doi.org/10.3389/fcimb.2022.873989 LaSarre, B., & Federle, M. J. (2013). Exploiting quorum sensing to confuse bacterial pathogens. Microbiology and molecular biology reviews, 77(1), 73-111. Leadbetter, J. R., & Greenberg, E. P. (2000). Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. Journal of bacteriology, 182(24), 6921-6926. Lee, S. J., Park, S. Y., Lee, J. J., Yum, D. Y., Koo, B. T., & Lee, J. K. (2002). Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Applied and environmental microbiology, 68(8), 3919–3924. https://doi.org/10.1128/AEM.68.8.3919-3924.2002 Li, M., Zhang, J. Z., Song, X. Y., Wang, Y., & Bie, S. T. (2011, May). Notice of Retraction: Effect of pH and temperature on activity of immobilized lactonase. In 2011 5th International Conference on Bioinformatics and Biomedical Engineering (pp. 1-4). IEEE. Luján Roca, D. Á. (2014). Pseudomonas aeruginosa: un adversario peligroso. Acta bioquímica clínica latinoamericana, 48(4), 465-474. http://www.scielo.org.ar/scielo.php?pid=S0325-29572014000400009&script=sci_arttext&tlng=pt M Florez, A., González, A., Pedroza, C. J., Correa, E., Rueda, N. J., & Orduz, S. (2014). Identification, cloning and lactonase activity of recombinant protein of N-acyl homoserine lactonase (AiiA) from Bacillus thuringiensis 147-115-16 strain. Revista Colombiana de Biotecnología, 16(1), 153-162. Mahan, K., Martinmaki, R., Larus, I., Sikdar, R., Dunitz, J., & Elias, M. (2020). Effects of signal disruption depends on the substrate preference of the lactonase. Frontiers in Microbiology, 10, 3003.. McCarthy, R. R., & O'Gara, F. (2015). The impact of phytochemicals present in the diet on microbial signalling in the human gut. Journal of Functional Foods, 14, 684-691. Miranda, S. W., Asfahl, K. L., Dandekar, A. A., & Greenberg, E. P. (2022). Pseudomonas aeruginosa Quorum Sensing. Advances in experimental medicine and biology, 1386, 95–115. https://doi.org/10.1007/978-3-031-08491-1_4 Morohoshi, T., Kamimura, Y., & Someya, N. (2020). Identification and Characterization of Quorum-Quenching Activity of N-Acylhomoserine Lactonase from Coagulase-Negative Staphylococci. Antibiotics (Basel, Switzerland), 9(8), 483. https://doi.org/10.3390/antibiotics9080483 Murugayah SA, Gerth ML. Engineering quorum quenching enzymes: progress and perspectives. Biochem Soc Trans. 2019 Jun 28;47(3):793-800. doi: 10.1042/BST20180165. Epub 2019 May 7. PMID: 31064863; PMCID: PMC6599154. Nain, Z., Adhikari, U. K., Abdulla, F., Hossain, N., Barman, N. C., Mansur, F. J., ... & Karim, M. M. (2020). Computational prediction of active sites and ligands in different AHL quorum quenching lactonases and acylases. Journal of biosciences, 45, 1-19. Nealson, K. H., Platt, T., & Hastings, J. W. (1970). Cellular control of the synthesis and activity of the bacterial luminescent system. Journal of bacteriology, 104(1), 313-322. New England Biolabs Canada & New England Biolabs. (s. f.). Ligation Protocol with T4 DNA Ligase (M0202). NEW ENGLAND Biolabs. https://www.neb.com/en/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202 New England Biolabs Canada & New England Biolabs. (s. f.). NEBExpress® ni Spin Columns | NEB. https://www.neb.ca/s1427 Oller Ruiz, I. N. (2023). Enfoque “One health” frente a la problemática de la resistencia a antibióticos. Organización Mundial de la Salud (OMS). (2022, 9 diciembre). Aumento de la resistencia a los antibióticos en infecciones bacterianas que afectan al ser humano. OMS. https://www.who.int/es/news/item/09-12-2022-report-signals-increasing-resistance-to-antibiotics-in-bacterial-infections-in-humans-and-need-for-better-data Organización Mundial de la Salud: OMS. (2021). La escasez mundial de antibióticos innovadores favorece la aparición y propagación de la farmacorresistencia. Organización Mundial de la Salud. https://www.who.int/es/news/item/15-04-2021-global-shortage-of-innovative-antibiotics-fuels-emergence-and-spread-of-drug-resistance Papenfort, K., Bassler, B. Quorum sensing signal–response systems in Gram-negative bacteria. Nat Rev Microbiol 14, 576–588 (2016). https://doi.org/10.1038/nrmicro.2016.89 Pedroza Padilla, C. J. (2010). Caracterización de la enzima acil homoserina lactonasa de una cepa de Bacilus thuringiensis (Doctoral dissertation). Pedroza, CJ, Flórez, AM, Ruiz, OS, & Orduz, S. (2014). Hidrólisis enzimática de moléculas asociadas con el quórum sensing bacteriano utilizando una acil homoserina lactonasa de una nueva cepa de Bacillus thuringiensis. Antonie Van Leeuwenhoek , 105 , 253-264. Prashanth, K., Vasanth, T., Saranathan, R., Makki, A. R., & Pagal, S. (2012). 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Frontiers in Microbiology, 13, 977673. Rémy, B., Plener, L., Decloquement, P., Armstrong, N., Elias, M., Daudé, D., & Chabrière, É. (2020). Lactonase specificity is key to quorum quenching in Pseudomonas aeruginosa. Frontiers in Microbiology, 11, 762. https://www.frontiersin.org/articles/10.3389/fmicb.2020.00762/full Rodríguez Tamayo, E. A., & Jiménez Quiceno, J. N. (2023). Resistencia bacteriana a antibióticos en ambientes acuáticos: origen e implicaciones para la salud pública. Rojas Badía, M. M. (2011). Quorum sensing en la asociación beneficiosa de las bacterias con las plantas. Revista colombiana de biotecnología, 13(2), 135-143. Rueda forero, n. J. (2016). Mejoramiento genético por “error prone pcr” del gen aiia que codifica para n- acíl homoserina lactonasa de bacillus thuringiensis. Universidad industrial de santander. Ryu DH, Lee SW, Mikolaityte V, Kim YW, Jeong HY, Lee SJ, Lee CH, Lee JK. Identification of a Second Type of AHL-lactonase from Rhodococcus sp. BH4, belonging to the α/β Hydrolase Superfamily. J Microbiol Biotechnol. 2020 Jun 28;30(6):937-945. doi: 10.4014/jmb.2001.01006. PMID: 32160697; PMCID: PMC9728292. Salinas, Y. R., Salinas, Y. M. R., & Wong, M. M. M. (2022). Enfoque social de la repercusión de la resistencia antimicrobiana y su control. Sambrook, J., & Russell, D. W. (2006). SDS-polyacrylamide gel electrophoresis of proteins. CSH Protoc, 1(4). See-Too, W. S., Convey, P., Pearce, D. A., & Chan, K. G. (2018). Characterization of a novel N-acylhomoserine lactonase, AidP, from Antarctic Planococcus sp. Microbial cell factories, 17, 1-14. Shah, M., Taylor, V. L., Bona, D., Tsao, Y., Stanley, S. Y., Pimentel-Elardo, S. M., McCallum, M., Bondy-Denomy, J., Howell, P. L., Nodwell, J. R., Davidson, A. R., Moraes, T. F., & Maxwell, K. L. (2021). A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa. Molecular cell, 81(3), 571–583.e6. https://doi.org/10.1016/j.molcel.2020.12.011 Sikdar, R., & Elias, M. (2020). Quorum quenching enzymes and their effects on virulence, biofilm, and microbiomes: a review of recent advances. Expert review of anti-infective therapy, 18(12), 1221–1233. https://doi.org/10.1080/14787210.2020.1794815 Studer, R. A., Dessailly, B. H., & Orengo, C. A. (2013). Residue mutations and their impact on protein structure and function: detecting beneficial and pathogenic changes. Biochemical journal, 449(3), 581-594. Suryanti, E., Rusmana, I., Wahyudi, A. T., & Akhdiya, A. (2020). Characterization and expression of acyl homoserine lactone (AHL) lactonase in E. coli BL21 (DE3) pLysS. EurAsian Journal of Biosciences, 14(2). Turovskiy, Y., Kashtanov, D., Paskhover, B., & Chikindas, M. L. (2007). Quorum sensing: fact, fiction, and everything in between. Advances in applied microbiology, 62, 191–234. https://doi.org/10.1016/S0065-2164(07)62007-3 V, S., Girija, A. S., Himabindu, Krishnan, M., & Babu, S. (2023). Anti-quorum sensing activity of Boerhavia diffusa against Pseudomonas aeruginosa PAO1. Bioinformation, 19(3), 310–318. https://doi.org/10.6026/97320630019310 Valerga, D. M., & Trombetta, L. (2022). Automedicación con antibióticos y resistencia bacteriana. Revista de la Asociación Médica Argentina, 135(3). Wang, L. H., Weng, L. X., Dong, Y. H., & Zhang, L. H. (2004). Specificity and enzyme kinetics of the quorum-quenching N-acyl homoserine lactone lactonase (AHL-lactonase). Journal of Biological Chemistry, 279(14), 13645-13651. Xie, Y., Chen, J., Wang, B., Peng, A. Y., Mao, Z. W., & Xia, W. (2022). Inhibition of Quorum-Sensing Regulator from Pseudomonas aeruginosa Using a Flavone Derivative. Molecules (Basel, Switzerland), 27(8), 2439. https://doi.org/10.3390/molecules27082439 Yada, S., Kamalesh, B., Sonwane, S., Guptha, I., & Swetha, R. K. (2015). Quorum sensing inhibition, relevance to periodontics. Journal of international oral health : JIOH, 7(1), 67–69. Zhang, Y., J. An, W. Ye, G. Yang, Z. G. Qian, H. F. Chen, L. Cui and Y. Feng (2012). "Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides." Appl Environ Microbiol78(18): 6647-6655. Zhuang, M., Achmon, Y., Cao, Y., Liang, X., Chen, L., Wang, H., Siame, B. A., & Leung, K. Y. (2021). Distribution of antibiotic resistance genes in the environment. Environmental pollution (Barking, Essex : 1987), 285, 117402. https://doi.org/10.1016/j.envpol.2021.117402https://doi.org/10.1016/j.envpol.2021.117402 |
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Suárez-Barera, Miguel Orlandof6a2ab36-c2e7-4d71-844a-e4f1c46f9d31-1Pérez-Vergel, Astrid Carolina9a846e04-456c-4429-9f51-d194b02e7fe2-1Valdivieso-Quintero, Wilfredob09a9bf7-eb04-416b-8fb6-82c7940c9f71-1Hernández-Peñaranda, Indira Paola54a8dcd7-6596-43da-9095-3de244aa8e67-1Laboratorio de Biología Molecular y Biotecnología2025-01-31T21:04:23Z2025-12-102025-01-31T21:04:23Z2024-11-28DigitalLa resistencia a los antibióticos es uno de los problemas más importantes en que hay en la salud humana la cual ha incrementado los costos médicos, la estancia hospitalaria y la mortalidad (Jiménez, 2020). Por este motivo la inhibición del QS se ha considerado como un enfoque terapéutico atractivo, para prevenir los fenotipos resistentes y virulentos debido a que es más específica e impone una menor presión selectiva hacía el patógeno así disminuyendo la probabilidad de que la bacteria genere resistencia (Espinoza & Esparza., 2021).Antibiotic resistance is one of the most important problems in human health, which has increased medical costs, hospital stays, and mortality (Jiménez, 2020). For this reason, QS inhibition has been considered an attractive therapeutic approach to prevent resistant and virulent phenotypes because it is more specific and imposes less selective pressure on the pathogen, thus decreasing the probability that the bacteria will generate resistance (Espinoza & Esparza., 2021).PregradoMicrobiólogo IndustrialUniversidad de SantanderIntroducción .................................................................................................................................. 19 Planteamiento del Problema ......................................................................................................... 22 Justificación .................................................................................................................................. 26 Hipótesis ....................................................................................................................................... 29 Hipótesis Alternativa .................................................................................................................... 29 Hipótesis Nula............................................................................................................................... 29 Marco Teórico............................................................................................................................... 30 Resistencia a los Antibióticos....................................................................................................... 30 Sistema de Comunicación Celular: Quórum Sensing (QS).......................................................... 31 Quorum Sensing en Bacterias Gram Negativas............................................................................ 32 Mecanismos de Inhibición del QS ................................................................................................ 37 AHL- Lactonasas.......................................................................................................................... 39 Mejoramiento de la Actividad Enzimática de las AHL-Lactonasas por Medio de la Formación de Mutantes........................................................................................................................................ 40 Estado del Arte.............................................................................................................................. 42 Objetivos....................................................................................................................................... 47 Objetivo General........................................................................................................................... 47 Objetivos Específicos.................................................................................................................... 47 Materiales y Métodos.................................................................................................................... 48 Diseño del Estudio ........................................................................................................................ 48 Cepas Utilizadas y Condiciones de Crecimiento .......................................................................... 48 Producción de la Lactonasa Variante en E. coli DE3BL21 (pCOLD IV) .................................... 49 Expresión y Purificación de la Enzima AHL-Lactonasa .............................................................. 51 Evaluación de la Actividad Inhibitoria de la AHL-Lactonasa Recombinante Frente al Biosensor CV026 a Diferentes Temperaturas y pH....................................................................................... 51 Cuantificación en Placa de la Producción de Violaceína.............................................................. 53 Evaluación de la Actividad Inhibitoria de la AHL-Lactonasa Recombinante Frente al Biosensor A. tumefaciens NTL4 a Diferentes Temperaturas y pH................................................................ 53 Análisis de Datos .......................................................................................................................... 54 Implicaciones Éticas ..................................................................................................................... 54 Resultados..................................................................................................................................... 57 Producción de la Lactonasa Epp9 en E. coli DE3BL21 (pCOLD IV).......................................... 57 Expresión y Purificación de la Enzima AHL-Lactonasa Recombinante ...................................... 58 Evaluación de la Actividad Inhibitoria de la AHL-Lactonasa Recombinante Frente al Biosensor CV026 a Diferentes Temperaturas y pH....................................................................................... 60 Determinación de la Actividad Inhibitoria de la AHL-Lactonasa Recombinante Frente NTL4 en Distintas Condiciones Temperaturas y pH ................................................................................... 69 Discusión....................................................................................................................................... 72 Conclusiones................................................................................................................................. 77 Recomendaciones ......................................................................................................................... 78 Referencias Bibliográficas............................................................................................................ 7989 papplication/pdfapplication/mswordUniversidad de SantanderT 33.24 P272cRepositorio Digital Universidad de 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