Using chitinolytic materials to obtain bioactive oligosaccharides: Characterization of biocatalysts

• Autores: Peter Elias Kidibule
• Directores de la Tesis: María Fernández Lobato (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2021
• Idioma: inglés
• Número de páginas: 181
• Títulos paralelos:
  • Utilización de materiales quitinolíticos para la obtención de oligosacáridos bioactivos: Caracterización de biocatalizadores y productos
• Tribunal Calificador de la Tesis: Francisco José Plou Gasca (presid.), Miguel Angel Rodriguez (secret.), Maria Camilla Baratto (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Chitin, the most abundant natural biopolymer in aquatic environment, is composed of repeating β-1-4 linked N-acetyl-β-D-glucosamine (GlcNAc) units, and constitutes the structural component of protective biological structures such as fungal cell walls, crustaceans and insect exoskeletons. Recently, enzymes acting on chitin and chitosan (a deacetylation product of chitin) have gained strong interest due to wide range of biotechnological applications of these polymer derivatives. The biological activity of chitin, chitosan and/ particularly chitooligosaccharides (COS) has made these compounds the targets for the industrial and medical sectors. In this context chitinases and chitosanases are essential glycosyl hydrolases (GH) for the biotransformation of chitin and chitosan into COS by hydrolyzing the β-1,4-linkages.

    In this work, two fungal chitinases, the endo-chitinase Chit33 and the exo-chitinase Chit42, both from Trichoderma harzianum were expressed in Pichia pastoris, using fed-batch fermentation, to 0.63 and 3 g/L, respectively. The heterologous proteins were purified, characterized biochemically and their kinetic parameters on different types of chitinolytic materials analyzed. Numerous variants of both enzymes were also obtained using rational targeted mutation based on their structural determinants. Utilization of the two enzymes (Chit42 also immobilized on magnetic nanoparticles and chitosan beads) as well as that of commercial preparations based on Bacillus spp cells extracts were analyzed for their application in the production of fully acetylated (faCOS), fully deacetylated (fdCOS) and partially acetylated (paCOS) COS mixtures. The product profiles were analyzed, when possible characterized using different chromatographic techniques and MALDI-TOF mass spectrometry, and their biological activity evaluated. COS mixtures in the range of 0.5-2 and 2-10 kDa produced from colloidal chitin and chitosan clearly exhibited antioxidant activity. The mixtures of fdCOS and faCOS apparently showed anti-inflammatory activity and some of them even a neuroprotective effect. The biological properties of COS obtained from chitin and chitosan gives biotechnological potential to both, the biocatalyst producing them and the products that they generate