Sintesís de oligosacáridos y glicoconjugados bioactivos empleando enzimas glicosídicas
EntityUAM. Departamento de Biología Molecular
Funded byGracias a todas las instituciones que han posibilitado, con su financiación, la realización de esta Tesis Doctoral. Gracias a los proyectos Nacionales BIO2013-48779-C4-1-R y BIO2016-76601-C3-1-R
SubjectsOligosacáridos - Síntesis - Tesis doctorales; Glicoconjugados - Síntesis - Tesis doctorales; Biología y Biomedicina / Biología
NoteTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 29-11-2019
Esta tesis tiene embargado el acceso al texto completo hasta el 29-05-2021
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Bioactive carbohydrates and glycoconjugates possess many biological activities that can exert a beneficial effect in human health. This Thesis is focused on the enzymatic production and characterization of bioactive glycoconjugates: fructosylated derivatives of hydroquinone and hydroxytyrosol; and three families of bioactive oligosaccharides: neofructooligosaccharides (neo-FOS), acidic xylooligosaccharides (acidic XOS) and chitooligosaccharides (COS). Neo-FOS production was investigated employing immobilized biocatalysts prepared by ionic binding or entrapment of the β-fructofuranosidase from Xanthophyllomyces dendrorhous (pXd-INV). The enzyme pXd-INV was entrapped in polyvinyl alcohol lenses and neo-FOS production was studied in a batch stirred tank reactor, obtaining a maximum productivity of 42 gFOS L-1 day -1. Neo-FOS synthesis was also assessed in a packed-bed bioreactor employing pXd-INV immobilized on an amino activated carrier (Sepabeads EC-HA) with an initial productivity of 218 gFOS L-1 day-1. The ability of pXd-INV to fructosylate phenolic compounds was also evaluated. Two positive acceptors were found: hydroxytyrosol (HT) and hydroquinone (HQ). The production of HT fructosides was optimized, yielding up to 11.7 g/L. The mechanism and selectivity of the reaction, and the molecular features that determine that a phenolic compound acts as an acceptor or an inhibitor, were studied by crystallography. The synthesis of acidic XOS was carried out with the commercial enzyme Depol 670L. Xylan hydrolysis gave rise to a mixture of neutral and acidic XOS. Acidic XOS were separated by anion-exchange chromatography and the main product was chemically characterized and identified as 2’-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). The antioxidant activity of the obtained product, neutral XOS and xylan was comparatively measured. Both X2_MeGlcA and xylan displayed scavenging activity on the ABTS radical cation. Furthermore, an integrated methodology was developed for the enzymatic production and characterization of several COS mixtures. A proteolytic preparation (Neutrase 0.8L) and a chitinase from Trichoderma harzanium (CHIT42) were employed for the hydrolysis of chitosans with different deacetylation degrees and molecular weights. Thus, we synthesized several COS mixtures based on: (1) fully deacetylated COS (fdCOS); (2) partially acetylated COS (paCOS); (3) mixtures of fdCOS and paCOS.
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Procesos biocatalíticos para la producción sostenible de oligosacáridos prebióticos empleando glicosiltransferasas inmovilizadas Fernández Arrojo, Lucía