176 research outputs found
Biotechnological platforms for aryl-alcohol oxidases by directed evolution
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 03-10-2019Esta tesis tiene embargado el acceso al texto completo hasta el 03-04-2021The aryl-alcohol oxidase (AAO) is a fungal flavoenzyme that supplies H2O2 to the ligninolytic consortium during natural wood decay. Being active on a wide array of aromatic alcohols, this GMC oxidase presents a highly enantioselective mechanism of great interest in organic synthesis processes. The most powerful strategy for the AAO to meet industrial standards is the engineering of its properties by directed evolution. In the present Doctoral Thesis, an evolutionary platform for the AAO from Pleurotus eryngii was developed in order to: (i) obtain functional expression in yeasts, (ii) design a secondary benzyl-alcohol oxidase, and (iii) explore the enzymatic conversion of furfural derivatives. To achieve functional expression in Saccharomyces cerevisiae, the AAO gene was fused to different signal peptides including chimeric versions of the mating-α factor and the killer K1 toxin preprosequences. The platform for in vitro evolution was completed with a dual high-throughput screening assay to detect H2O2 that included a method based on the Fenton reaction. To enhance secretion, several libraries were created combining classical evolution (i.e. mutagenic PCR and DNA shuffling) with structure-guided evolution by MORPHING. The final secretion variant FX9, carried four mutations in the signal peptide and two substitutions in the mature protein including the consensus/ancestral H91N. The FX9 improved secretion up to 4.5 mg/L and presented high stability and kinetic values similar to the native enzyme. FX9 was cloned and expressed in Pichia pastoris maintaining expression levels and main biochemical properties. When the production was scaled-up in 5L fermenter, AAO production was increased to 25.5 mg/L. FX9 was further evolved to selectively oxidize secondary benzyl alcohols. The residual activity on chiral molecules was unlocked with the modulation for the catalytic pocket by combinatorial saturation mutagenesis. After four generations, that included a site-directed recombination step to polish mutations, LanDo variant harbored five new substitutions increasing the catalytic efficiency with 1-(p-methoxyphenyl)-ethanol in 3 orders of magnitude with a 99% ee. Exploring the transformation of 5-hydroxymethylfurfural (HMF) into furan-2,5-dicarboxylic acid (FDCA), FX9 acquired mutation F501W that improved catalytic efficiency on HMF 3-fold and showed for the first time the performance of three consecutive oxidations for the AAOLa aril-alcohol oxidasa (AAO) es una flavoenzima fúngica que suple H2O2 al consorcio ligninolítico durante la degradación natural de la madera. Siendo activa con una amplia variedad de alcoholes aromáticos, esta oxidasa GMC presenta un mecanismo altamente enantioselectivo de gran interés en procesos de síntesis orgánica. La estrategia más potente para adaptar a la AAO a estándares industriales es la ingeniería de sus propiedades mediante técnicas de evolución dirigida. En la presente Tesis Doctoral, una plataforma evolutiva para la AAO de Pleurotus eryngii fue desarrollada con el objetivo de: (i) obtener expresión funcional en levaduras, (ii) diseñar una aril-alcohol oxidasa activa con alcoholes secundarios, y (iii) explorar la conversión enzimática de derivados del furfural. Para obtener expresión funcional en Saccharomyces cerevisiae, el gen de la AAO se fusionó a diferentes péptidos señales incluyendo versiones quiméricas de las secuencias prepro del factor-α y la toxina killer K1. La plataforma para la evolución in vitro se completó con un ensayo dual de screening para la detección de H2O2 incluyendo un método basado en la reacción de Fenton. Para mejorar la secreción, se crearon varias librerías combinando evolución clásica (PCR mutagénica y DNA shuffling) con evolución focalizada con el método MORPHING. La variante final FX9, con alta estabilidad y constantes cinéticas similares a la enzima nativa, presentó cuatro mutaciones en el péptido señal y dos substituciones en la proteína madura incluyendo la consenso/ancestral H91N. FX9 se expresó en S. cerevisiae con valores de 4.5 mg/L y fue posteriormente clonada y expresada en Pichia pastoris a escala de fermentador de 5 L alcanzando niveles de secreción 25.5 mg/L y manteniendo sus propiedades bioquímicas generales. La variante FX9 fue sometida a posteriores ciclos de evolución, incluyendo el remodelado del bolsillo catalítico por mutagénesis saturada combinatorial, para la oxidación de alcoholes bencílicos secundarios. Las cinco mutaciones introducidas en la variante LanDo aumentaron la eficiencia catalítica con 1-(p-methoxyphenyl)-ethanol en 3 órdenes de magnitud con un 99 % ee. Explorando la transformación del 5-hydroxymethylfurfural (HMF) en furan-2,5-dicarboxylic acid (FDCA), FX9 adquirió la mutación F501W que mejoró 3 veces la eficiencia catalítica con HMF y demostró por primera vez la catálisis de 3 oxidaciones consecutivas para la AAOLa financiación que me ha permitido seguir mis estudios doctorales. Los proyectos europeos “Optimized oxidoreductases for medium and large scale industrial biotransformations (INDOX FP7-KBBE-2013-7-613549)” y “New enzymatic oxidation/oxyfunctionalization technologies for added value bio-based products. (ENZOX2 H2020-BBI-PPP-2015-2-720297)”. Los proyectos nacionales “Evolución dirigida de oxidoreductasas ligninolíticas modernas y ancestrales para el diseño de una levadura de podredumbre blanca (DEWRY BIO2013-43407-R)”, “Evolución dirigida y computacional de ligninasas (LIGNOLUTION BIO2016-79106-R)“ y “Química sintética mediante enzimas quiméricas de fusión diseñadas por evolución dirigida y computacional (EVOCHIMERA Y2018/BIO-4738)”
Reacción de Diels-Alder intramolecular organocatalizada usando el anillo de furano como dieno (IMDAF)
Este trabajo fin de grado tiene como objetivo llevar a cabo reacciones enantioselectivas intramoleculares de Diels-Alder empleando el anillo de furano como dieno (conocidas como reacciones IMDAF). Con este fin, se ha llevado a cabo la síntesis de derivados en los que el furano queda unido al filodieno mediante una cadena hidrocarbonada simple (C) o conteniendo un heteroátomo (N, O). En el extremo de dicha cadena siempre se instala un grupo extractor de carga (EWG) para favorecer dicha reacción. Para realizar la cicloadición con estos derivados se han empleando una serie de organocatalizadores que permiten el control de la enantioselectividad del cicloaducto formado.Grado en Químic
La crisis de la televisión pública: el caso de RTVV y los retos de una nueva gobernanza
Javier Marzal Felici, Jessica Izquierdo Castillo y Andreu Casero Ripollés (eds.) Aldea Global, Barcelona, Castelló de la Plana y Valencia, 2015Javier Marzal Felici, Jessica Izquierdo Castillo y Andreu Casero Ripollés (eds.) Aldea Global, Barcelona, Castelló de la Plana y Valencia, 201
La crisis de la televisión pública: el caso de RTVV y los retos de una nueva gobernanza. Javier Marzal Felici, Jessica Izquierdo Castillo y Andreu Casero Ripollés (eds.) Aldea Global, Barcelona, Castelló de la Plana y Valencia, 2015. 272 páginas
Obra ressenyada: Javier MARZAL FELICI, Jessica IZQUIERDO CASTILLO y Andreu CASERO RIPOLLÉS (eds.), La crisis de la televisión pública: el caso de RTVV y los retos de una nueva gobernanza. Barcelona; Castelló de la Plana y Valencia: Aldea Global, 2015
Exercise redox biology from health to performance
In order to support the energy demand during physical exercise, temporary acute responses occur in our organism to meet the homeostatic challenge imposed by the working muscles. As a result of the accumulation of exercise bouts the organism adapts. Exercise adaptations refer to the long-term changes that occur in our body as a consequence of training. The molecular bases of skeletal muscle adaptations to exercise such as mitochondrial biogenesis, hypertrophy, angiogenesis, and cytoprotection, are mediated by signaling events regulating transcriptional and translational processes and the activity of proteins involved in the maintenance of homeostasi
Los jóvenes españoles demandan una televisión con más ficción y entretenimiento
El actual panorama mediático se ha visto afectado en los últimos años por un proceso de evolución hacia el modelo digital que ha producido un importante cambio sociológico con vocación de afectar de lleno a la cultura audiovisual. Las nuevas generaciones han transformado su modo de consumir productos audiovisuales, acelerando una tendencia ‘one to one’, individualista y de consumo rápido, que pasa necesaria e irremediablemente por Internet. Este artículo parte de una investigación que tiene como objetivo principal la cultura de ocio y entretenimiento de los jóvenes (14-25 años) que, en este caso, centramos en la televisión.The current media state has been influenced last years by a process of evolution towards the digital model. As a consequence, an important sociological transformation has occurred with the aim of affecting entirely the media culture in a short space of time. New generations have change the way of consuming media products, speeding up a ‘one to one’ tendency (individualistic and fast consumerist) that goes necessary and inevitably through the Net of networks. This article starts off an investigation whose main aim is leisure culture and entertainment for the young (14-25 years) that, in this case, is focused on television
Composición en isótopos estables (18O y Deuterio)y silicio disuelto del agua de lluvia, de manantiales y de río en la cuenca de cabecera del Río Landro (Lugo)
Hornos de brea en Tenerife. Identificación y catalogación
La producción de pez o brea tuvo una gran importancia en la economía tinerfeña desde
fines del siglo XV, como recoge la documentación de la época. En este trabajo realizamos
una aproximación a los yacimientos arqueológicos de los hornos de brea, en los siglos XVI y
XVII, mediante su catalogación e identificación en las distintas comarcas históricas del espacio
insular. Estos hornos, aparte de su valor patrimonial, constituyen un indicador de la
ubicación de las masas forestales, de las vías de tránsito o de la dirección de las primeras
producciones de brea en la isla de Tenerife.The production of pitch or tar was of great importance to the Tenerife economy from the
latter part of the XV century, as can be derived from the documentation of the period. In
this work we approach the archaeological strata of the pitch ovens in the XVI and XVII centuries
by cataloguing and identifying them in the various historical territorial divisions of the
island. In addition to their heritage value these ovens are tracers of the locations of the
afforested areas, of the traffic routes, and of the earliest sites where pitch was produced in
the Island of Tenerife
Switching the substrate preference of fungal aryl-alcohol oxidase: towards stereoselective oxidation of secondary benzyl alcohols
Oxidation of primary alcohols by aryl-alcohol oxidase (AAO), a flavoenzyme that provides H2O2 to fungal peroxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary aryl-alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.This work was supported by the INDOX (KBBE-2013-7-613549) EU project and by the BIO2017-86559-R (GenoBioref), CTQ2016-79138-R and BIO2016-79106-R projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds. Pedro Merino (University of Zaragoza, Spain) is acknowledged for his suggestions on chiral HPLC analyses.Peer ReviewedAward-winningPostprint (published version
QSAR for Anti-RNA-Virus Activity, Synthesis, and Assay of Anti-RSV Carbonucleosides Given an Unify Representation of Spectraö Moments, Quadratic, and Topologic Indices
The 9th International Electronic Conference on Synthetic Organic Chemistry session Computational ChemistryThe unify representation of spectral moments, classic topologic indices, quadratic indices, and stochastic molecular descriptors shown that all these molecular descriptors lie within the same family. Consequently, the same priori probability for a success quantitative-structure-activity-relationship (QSAR) may be expected no matter which indices are selected. Herein, we used stochastic spectral moments as molecular descriptors to seek a QSAR using a database of 221 bioactive compounds previously tested against diverse RNA-viruses and 402 non-active ones. The QSAR model thus obtained correctly classifies 90.9 % of compounds in training. The model also correctly classifies a total of 87.9 % of 207 compounds on additional external predicting series, 73 of them having anti-RNA-virus activity and 134 non-active ones. In addition, all compounds were regrouped into five different subsets for leave-group-out studies: 1) antiinfluenza, 2) anti-picornavirus, 3) anti-paramyxovirus, 4) anti-RSV/anti-influenza, and 5) broad range anti-RNA-virus activity. The model has retained overall accuracies about 90 % on these studies validating model robustness. Finally, we exemplify the practical use of the model with the discovery of compounds 124 and 128. These compounds presented MIC50 values = 3.2 and 8 µg/mL against respiratory syncytial virus (RSV) respectively. Both compounds have also low cytotoxicity expressed by their Minimal Cytotoxic Concetrations > 400 µg/mL for HeLa cells. The present approach represent and effort toward a formalization and application of molecular indices in bioinformatics, bioorganic and medicinal chemistryAuthors would like to express their gratitude by partial financial support to the Department of Organic Chemistry, University of Santiago de Compostel
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