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 mu M 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 mgL-1h(-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.