Development of a flow microreactor with immobilized catechol-1,2-dioxygenase for the biocatalytic production of cis,cis-muconic acid

A low cost microreactor was developed by stable immobilization of catechol-1,2-dioxygenase for the flow biocatalysis of catechol to produce cis,cis-muconic acid, a direct precursor of industrially relevant adipic acid. The microreactors of polydimethylsiloxane were manufactured with different designs by straightforward, cost-effective soft lithography techniques, and were tested with different immobilization strategies focused on preserving enzyme catalytic activity and stability over time. The best immobilization strategy was covalent immobilization by amino-silanization followed by glutaraldehyde linker grafting. The biocatalytic activity of the immobilized enzyme was assessed by measuring muconic acid production through the flow of 50 µM catechol at room temperature. Catechol conversion rates of up to 95 % were achieved over a 5-hour period, and volumes of up to 180 mL were processed using only 0.32 nmol of immobilized protein, resulting in the production of 1.33 mg·L⁻¹·h⁻¹. This work demonstrates the feasibility of using microreactor-based flow biocatalysis for sustainable muconic acid production and highlights its promising application at industrial scale.