Chloroperoxidase and D-fructose-6-phosphate aldolase in enzymatic cascade reactions for the synthesis of iminocyclitols /

• Autores: Gerard Masdeu Gamez
• Directores de la Tesis: Josep López Santín (dir. tes.), Gregorio Alvaro Campos (codir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2017
• Idioma: español
• Tribunal Calificador de la Tesis: Marielle Lemaire (presid.), Francisco Valero Barranco (secret.), Carmen López Díaz (voc.)
• Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad Autónoma de Barcelona
• Materias:
  • Física
    • Química física
      • Catálisis
    • Física del estado sólido
• Enlaces
  • Tesis en acceso abierto en:  DDD  TDX 
• Resumen

  • This thesis work is focused on the feasibility of a peroxidase/aldolase cascade reaction for the synthesis of iminocyclitols, which are glycosidase inhibitors with enormous therapeutic potential in many diseases by altering the glycosylation or catabolism of glycoproteins. The proposed system consists of two enzymes –chloroperoxidase (CPO) and D-fructose-6-phosphate aldolase (FSA)– for the formation of the precursor of D-fagomine, selected as the target iminocyclitol to evaluate the viability of the coupling of both reactions. The starting substrate is a β-amino alcohol (N-Cbz-3-aminopropanol, β-OH), which is oxidized to N-Cbz-3-aminopropanal (β-CHO) by CPO in presence of peroxide. FSA catalyzes the aldol addition of dihydroxyacetone to β-CHO to render the final product.

    Both reactions are investigated in order to understand the reaction mechanism, prepare suitable stabilized biocatalysts, and find the proper conditions for its application. The amino alcohol oxidative capacity of CPO regarding the substrate structure is discussed, followed by experimental trials on the β-OH oxidation to determine the main operating conditions for the first assays of the coupled reaction. However, the inactivation of both enzymes and the existence of side reactions, such as the further oxidation of β-CHO to N-Cbz-3-aminopropanoic acid (β-COOH) and a chemical reaction aldehyde-peroxide, are observed.

    Kinetic modeling of all the reactions represents an approach for a better understanding of the system and a possible strategy to optimize it. A mathematical model is systematically developed and validated. Concurrently, enzyme immobilization becomes a second strategy for the system improvement, by reducing the inactivation of CPO and FSA. The immobilization of both enzymes on functionalized conventional supports of agarose and magnetic nanoparticle clusters is studied.

    Finally, the kinetic model is employed for process intensification to determine the optimal conditions for the reaction coupling. Two reactor configurations are assayed: one-pot or two-consecutive vessels. Both strategies are compared using soluble and immobilized CPO/FSA.