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The aim of this study was to get insight into the internalization and transport of PEGylat-ed mixed micelles loaded by vitamin K, as mediated by Scavenger Receptor B1 (SR-B1) that is abundantly expressed by intestinal epithelium cells as well as by differentiated Caco-2 cells. Inhibition of SR-B1 reduced endocytosis and transport of vitamin-K-loaded 0%, 30% and 50% PEGylated mixed micelles and decreased colocalization of the micelles with SR-B1. Confocal fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis, and surface plasmon resonance (SPR) were used to study the interaction between the mixed micelles of different compositions (varying vitamin K loading and PEG content) and SR-B1. Interaction of PEGylated micelles was independent of the vitamin K content, indicating that the PEG shell prevented vitamin K exposure at the surface of the micelles and binding with the receptor and that the PEG took over the micelles’ ability to bind to the receptor. Molecular docking calculations corroborated the dual binding of both vita-min K and PEG with the binding domain of SR-B1. In conclusion, the improved colloidal stability of PEGylated mixed micelles did not compromise their cellular uptake and transport due to the affinity of PEG for SR-B1. SR-B1 is able to interact with PEGylated nanoparticles and mediates their subsequent internalization and transport.

Trabajo presentado en la 33ª ed. de la reunión científica regional "ZYMOMADRID" organizada por la Sra. Catedrática en Microbiología y Pasitología Dra. dña. María Molina Martín (Dpto. Microbiología II, Fac. Farmacia, UCM) en el Campus de Moncloa (UCM, Madrid, España) durante el día 18 de marzo de 2019., En la actualidad, la síntesis enzimática de productos específicos con un valor añadido está adquiriendo un mayor peso en la industria debido a su bajo coste y respeto hacia el medio ambiente. Por ello, la búsqueda de nuevas enzimas en levaduras no convencionales permite alternativas a los métodos químicos convencionales. La levadura Schwanniomyces occidentalis expresa una β-fructofuranosidasa (Ffase, EC 3.2.1.26) que, además de hidrolizar sacarosa, es capaz de transferir moléculas de fructosa al disacárido y generar pequeños fructooligosacáridos (FOS) con enlaces β-(2→6) y β-(2→1), básicamente 6-kestosa y 1-kestosa, respectivamente. La enzima puede fructosilar también distintos monosácaridos y compuestos hidroxilados, entre ellos polioles como el manitol y eritritol. La estructura 3D de la Ffase fue ya previamente caracterizada, se obtuvieron distintos complejos proteína-sustratos/productos y los determinantes estructurales implicados en la capacidad hidrolasa y transferasa de la proteína fueron también determinados. En este trabajo, se han analizado las bases moleculares de la selectividad del proceso de producción de fructosil-eritritol/manitol de Ffase. Para ello, se purificaron los correspondientes polioles fructosilados y se cristalizaron en complejo con la variante inactiva de la proteína Ffase-D50A, previamente expresada en Saccharomyces cerevisiae. El análisis cristalográfico de los complejos reveló que los residuos Q176 y Q228 de la proteína tenían un papel fundamental en el reconocimiento de manitol, y la posición N254 en el del eritritol. Se realizaron reacciones de fructosilación de polioles utilizando variantes mutantes de la Ffase en las que las posiciones referenciadas habían sido sustituidas por diferentes aminoácidos y se evaluaron los productos obtenidos utilizando técnicas cromatográficas (HPLC-ELSD). Los resultados verificaron el papel de estos residuos en el proceso de producción de polioles fructosilados, y en uno de los mutantes utilizados se logró mejorar la producción de fructosil-eritritol en un 20%.

Según diversos estudios epidemiológicos, la alimentación juega un papel determinante en la salud humana. Se ha demostrado que la dieta tiene un efecto directo sobre la composición y el equilibrio de la microbiota intestinal y, ésta, sobre la salud del individuo. Por ello, se buscan nuevos alimentos “funcionales”, como los oligosacáridos prebióticos, que son capaces de estimular el crecimiento de bacterias beneficiosas. Por otra parte, la glicosilación de distintos compuestos químicos mejora sus propiedades y su biodisponibilidad. Estos nuevos compuestos bioactivos aún no están tan desarrollados como los prebióticos, pero tienen una gran proyección de futuro en la industria farmacéutica. La investigación sobre la biosíntesis de estos productos conlleva tanto el estudio de nuevos compuestos con mejores propiedades funcionales como la búsqueda de formas más eficaces de producirlos. En este aspecto, la Biotecnología Enzimática se ha impuesto frente a otras metodologías de química tradicional gracias a su excelente regio-­‐, quimio-­‐ y estéreo-­‐especificidad. Y para profundizar en esta metodología, la Cristalografía de rayos X genera la información estructural básica para el diseño racional en la ingeniería de proteínas. Por todo ello, esta tesis se ha centrado en el estudio cristalográfico de glicosil hidrolasas (GH) que son las enzimas capaces de metabolizar carbohidratos, los biocompuestos estructuralmente más diversos en la naturaleza. Estas enzimas, altamente específicas y eficientes, intervienen en multitud de procesos biológicos esenciales por lo que un conocimiento detallado de su función a nivel molecular es clave para entender dichos procesos y controlar numerosas enfermedades, además de permitir optimizar su aplicación biotecnológica. En este manuscrito, se presentan cuatro trabajos de investigación realizados con enzimas productoras de carbohidratos prebióticos del tipo FOS (fructooligosacáridos), IMOS (isomaltooligosacáridos) o β-­‐glucanos, además de explorar su capacidad para sintetizar otros compuestos bioactivos. El objetivo general de la investigación es avanzar en el esclarecimiento de los mecanismos de reconocimiento proteína-­‐carbohidrato que modulan la especificidad de glicosidasas, mediante la Cristalografía de proteínas. Además de las implicaciones fundamentales para entender los procesos en los que estas enzimas intervienen, este conocimiento estructural es la base fundamental para realizar la ingeniería molecular de enzimas. El fin último es producir oligosacáridos con propiedades prebióticas, diseñados específicamente para manipular la microbiota intestinal del individuo, lo que abre la puerta a una futura medicina preventiva personalizada...

Trabajo presentado en la 7ª ed. del congreso internacional "FEMS" organizado por la Sociedad Española de Microbiología y la Federación Europea de Sociedades Microbiológicas en el Centro de Convenciones Feria Valencia (Valencia, España) durante los días 9 al 13 de julio de 2017., BACKGROUNDS: Chitooligosaccharides (COS) are β-(1,4)-linked oligomers of N-acetyl-glucosamine (GlcNAc) and glucosamine (GlcN) formed by chemical or enzymatic hydrolysis of chitosan or chitin. The growing biotechnological interest of COS in fields such as food or health increases the demand of the producing enzymes as well as their characterization and functional improvement. | OBJETIVES: Express a chitinase of 42 kDa from Trichoderma harzianum in a heterologous system, obtain protein levels compatible with its crystallization for the future protein structural resolution and evaluate the ability of the recombinant protein to produce COS. | METHODS: The chitinase gene cDNA from T. harzianum was expressed in Pichia pastoris using a restriction-free cloning strategy, production of heterologous protein was analysed and escalated up to a 5 L fermenter level. Recombinant protein was purified and some crystals were obtained which allows undertake the protein structural resolution. Synthesis of oligosaccharides from different substrates were evaluated and optimized using the recombinant enzyme. HPAEC-PAD on a Dionex ICS3000 system and Mass Spectrometry were used in the reaction studies and product characterization. | CONCLUSIONS: A chitinase of 42 kDa from T. harzianum was overexpressed in P. pastoris, the recombinant protein was purified, characterized and crystallized for the protein structural resolution. Production of COS mediated by this enzyme was evaluated and some of the molecules formed were characterized.

Trabajo presentado en el evento conjunto de la 12ª ed. del simposio internacional "Jornada de Carbohidratos" y la 3ª ed. de la reunión nacional "Grupo Especializado de Química Biológica" organizado por la Real Sociedad Española de Química en el Centro de Investigaciones Biológicas (CIB, CSIC) en Madrid (España) del 14 al 16 de marzo del año 2016., Enzymatic glycosylation of hydrophobic scaffolds can be catalyzed by glycosidases and glycosyltransferases. Glycosylation dramatically changes the physico-chemical properties of bioactive molecules, modulates their pharmacokinetic properties and influences their membrane transport. Polyphenols are, in general, hydrophobic compounds exhibiting poor absorption, which renders a very low bioavailability. The main objective of this work was to develop strategies for the enzymatic glycosylation of polyphenols, namely epigallocatechin gallate (EGCG, present in green tea), hesperetin (citrus fruits), resveratrol (grapes) and hydroquinone. The yield of glycoderivatives is usually low because the active-site architecture of glycosidic enzymes is not designed to accommodate non-natural acceptors −in contrast with the natural ones, i.e. carbohydrates−. In addition, the reactivity of phenolic groups is notably lower than the corresponding to typical hydroxyls. However, the synergistic combination of enzyme screening, medium engineering and structural analysis was employed to develop strategies for the glycosylation of different polyphenols. We have achieved the enzymatic synthesis of various α-glucosyl derivatives of resveratrol, EGCG or hesperetin by a transglycosylation reaction catalyzed by cyclodextrin glucanotransferase (EC 2.4.1.19, CGTase), a transglycosidase that employs easily available carbohydrates (e.g. starch or maltodextrins) as glucose donors. A common strategy to increase the yield of glycoderivatives was the use of cosolvents that favoured the solubilization of the phenolic acceptors. The antioxidant and surfactant properties of several of the synthesized compounds were evaluated. For the introduction of fructosyl groups, we found that the β-fructofuranosidase (EC 3.2.1.26) from the yeast Xanthophyllomyces dendrorhous was able to fructosylate hydroquinone. Interestingly, other polyphenols such as EGCG acted as strong inhibitors of β-fructofuranosidases from X. dendrorhous and Schwanniomyces occidentalis. The experimental results were validated by soaking the enzyme with the polyphenols followed by structural characterization of the complexes.

Trabajo presentado en el ANQUE.ICCE.BIOTEC - Congress on Chemistry, Chemical Engineering and Biotechnology, celebrado en Madrid del 01 al 04 de julio de 2014., The β-fructofuranosidase Xd-INV from Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) is an extracellular glycoprotein with a content of 59-67% N-linked carbohydrates and an estimated molecular mass of 160-200 kDa. This protein is included in family 32 of glycosylhydrolase (GH 32) on the basis of its amino acid sequence. Xd-INV, independently of the glycoforms, catalyzes the synthesis of neofructooligosaccharides (neoFOS), mainly neokestose and neonystose, sugars with enhanced prebiotic properties and higher chemical stability compared to traditional fructooligosaccharides (FOS). To the best our knowledge, this enzyme is the largest neoFOS microbial producer reported up to now [1]. Functionality of Xd-INV gene in Saccharomyces cerevisiae was previously reported [2], but only weak Xd-INV invertase activity was quantified in the cellular fraction of the transformant strain. In order to improve Xd-INV activity level, different heterologous expression systems, based in S. cerevisiae and Pichia pastoris, were evaluated. The DNA fragment encoding Xd-INV was fused to the S. cerevisiae MFα1 secretion signal and included in different expression vectors. The best expression level was obtained using P. pastoris and the produced enzyme was enzymatically characterized as well as analyzed on polyacrylamide gels using native or denaturing conditions and mass spectrometry. This study may provide the basis for a further mutational analysis of this enzyme that should shed additional light on the determinants responsible for fructosyltransferase activity within GH 32 enzyme family and the improvement of neoFOS production.

Trabajo presentado en la 4th International Conference on Novel Enzymes, celebrada en Gante (Bélgica) del 14 al 17 de octubre de 2014., Apart from sucrose hydrolysis, beta-fructofuranosidases (beta-FFases) also catalyze the transfer of the fructose moiety from sucrose to other carbohydrates (called acceptors, including sucrose itself), thus forming homo- and hetero-fructooligosaccharides (FOS) with potential applications in functional foods (prebiotics), pharmaceutical and cosmeceutical industries. Most fungal beta-FFases catalyze the formation of beta(2¿1) linkages between fructose units (inulin-type 1F-FOS, e.g. 1-kestose or nystose). We have isolated, purified, characterized and crystallized several extracellular ¿-fructofuranosidases from cultures of the yeasts Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma), Schwanniomyces occidentalis and Rhodotorula dairenensis with novel specificities. The ¿-FFase from X. dendrorhous is able to synthesize FOS of the 6G-series (neoFOS, e.g. neokestose and neonystose), which contain a ¿(2¿6) linkage between a fructose and the glucosyl moiety of sucrose.1 In addition, this enzyme fructosylates other disaccharides that contain glucose (maltose, isomaltulose, isomaltose, trehalose) and higher size oligosaccharides (maltotriose, raffinose, maltotetraose).2 S. occidentalis ¿-FFase forms 6F-FOS, which contain ¿(2¿6)-linked fructose units (with a levan-type structure; e.g. 6-kestose).3 The ¿-FFase from R. dairenensis synthesizes a complex mixture of 1F-FOS, 6F-FOS and 6G-FOS.4 The immobilization of several FFases has been also investigated to improve the corresponding bioprocesses.

© 2013 by The American Society for Biochemistry and Molecular Biology, Inc., Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv)and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Aring; resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition., This work was supported in part by Grants BIO2010-20508-C04-02 and BIO2010-20508-C04-03 from the Spanish Ministry of Education and Science.