Protocell-based continuous flow reactors for enzyme and whole-cell biocatalysis

Abstract

The design and construction of continuous flow biochemical reactors comprising immobilized biocatalysts have generated great interest in the efficient synthesis of value-added chemicals. Living cells use compartmentalization and reaction-diffusion processes for spatiotemporal regulation of biocatalytic reactions and implementing these strategies into continuous flow reactors could offer new opportunities in reactor design and application. Herein, we demonstrate the fabrication of protocell-based continuous flow reactors for enzyme and whole-cell mediated biocatalysis.
In the first two experimental chapters, we employ semipermeable membrane-bound coacervate vesicles as model protocells that spontaneously sequester enzymes or accumulate living bacteria to produce embodied microreactors capable of single- or multiple-step catalytic reactions. By packing millions of the enzyme/bacteria-containing coacervate vesicles in a glass column, we demonstrate a facile, cost-effective, and modular methodology capable of performing oxidoreductase, peroxidase and lipolytic reactions, enzyme-mediated L-DOPA synthesis and whole-cell glycolysis under continuous flow conditions. We show that the protocell-nested enzymes and bacterial cells exhibit enhanced activities and stability under deleterious operating conditions compared with their non-encapsulated counterparts.
In the third experimental chapter, we adopt the flow strategy from the blood circulatory system to develop a new continuous flow system for the application of microscale biocatalytic agents. DEAE-dextran/DNA microcapsules capable of enzyme immobilization are prepared as artificial blood cells. A liquid metal droplet is adopted as the system heart to induce fluid movement inside a closed-loop open-top channel device. Continuous flow biocatalysis with improved mass transfer conditions and spontaneous regeneration of enzyme cofactors NAD(P)H is achieved in the circular flow system.
These results provide a step towards the engineering of continuous flow reactors based on cell-like microscale agents and offer opportunities for the development of green and sustainable industrial bioprocessing.

Date of Award	1 Oct 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Stephen Mann (Supervisor) & Tom Oliver (Supervisor)