Síntesi asimètrica d'amines quirals mitjançant transaminases: un enfocament multienzimàtic a través de l'acoblament de piruvat decarboxilasa

• Autores: Natàlia Alcover Fortuny
• Directores de la Tesis: Gregorio Alvaro Campos (dir. tes.), Marina Guillén Montalbán (codir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2021
• Idioma: catalán
• ISBN: 9788449099557
• Tribunal Calificador de la Tesis: Francisco Valero Barranco (presid.), Pere Clapés Saborit (secret.), Marc Carnicer Heras (voc.)
• Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad Autónoma de Barcelona
• Enlaces
  • Tesis en acceso abierto en: TDX
• Resumen

  • The present thesis is focused on the development and optimization of a biocatalytical approach for the synthesis of chiral amines, which are highly valuable optically active compounds that can be used for the synthesis of numerous targets, especially in pharmaceutical and agrochemical industry. More specifically, 3-amino-1-phenylbutane (3-APB) and 1-phenylethylamine (1-PEA) synthesis is pretended by the cascade reaction of transaminase (TA) and pyruvate decarboxylase (PDC). The mentioned cascade consists in an asymmetric synthesis from their corresponding prochiral ketones and alanine catalyzed by omega-transaminase, which presents an unfavorable equilibrium. To overcome this problem, PDC acts as a by product removing system by transforming the resulting pyruvate to acetaldehyde and CO2, which leads to an equilibrium shift.

    Aiming to overcome the low PDC commercial availability, which can only be acquired at low amounts and a high cost, a whole production process was developed. Zymobacter palmae PDC (ZpPDC) gene was cloned and overexpressed in Escherichia coli. After that, high amounts of the recombinant enzyme were obtained by the development of a high-cell density culture process in bench-top bioreactor. Regarding TA, four different enzymes were available from Chromobacterium violaceum (Cvi-TA), Vibrio fluvialis (Vfl-TA) and Aspergillus terreus (Ate-TA and Ate-TA_T247S). Both PDC and the different transaminases were characterized to find out the appropriate compromise conditions to construct the enzymatic cascade. Taking into account the found conditions, preliminary screening reactions were carried out, from which Cvi-TA and Vfl-TA were selected for the synthesis of 3-APB; and Vfl-TA for the synthesis of 1-PEA.

    After proving the feasibility of TA and PDC cascade reaction, different optimization approaches were applied in order to maximize reaction yields and to improve the low transaminase operational stability. On the one hand, reaction conditions optimization approaches were explored. On the other, reaction medium engineering was applied. After that, enzyme immobilization was carried out. Immobilized derivatives of both Cvi-TA and Vfl-TA were obtained in MANA-agarose and epoxy-agarose supports. In the case of PDC, an innovative simultaneous purification and immobilization process was developed using MANA-agarose. Finally, the obtained immobilized enzymes were applied in reactions and a reaction cycle strategy was developed.