Immobilization of Alginate Microbeads With Whole-Cells Expressing Nitrilase for Continuous Nicotinic Acid Production in Chip Millireactor.

The biocatalytic production of nicotinic acid using nitrilases has emerged as a promising alternative to conventional chemical synthesis, offering improved selectivity and environmental sustainability. In this study, we developed a continuous-flow millireactor system employing Escherichia coli cells expressing a nitrilase from Paraburkholderia phymatum (NitPhym) for the continuous production of nicotinic acid. To enhance catalytic efficiency and stability, whole cells were immobilized using two different strategies: covalent attachment to the reactor walls and encapsulation in alginate microbeads. The performance of these immobilization techniques was evaluated in millireactors with two different geometries: pillar and zigzag configurations. Encapsulation of NitPhym-expressing E. coli cells in alginate microbeads was achieved through a centrifuge-assisted microencapsulation method, yielding beads with diameters of up to 350 µm. Continuous-flow reactions demonstrated that the microencapsulated cells maintained their activity over at least 7 days, achieving stable conversion rates of approximately 30% in both pillar and zigzag millireactors. Compared to direct covalent immobilization, microencapsulation significantly reduced cell leaching and contamination of the reaction product. These findings highlight the potential of whole-cell microencapsulation for enhancing the stability and efficiency of biocatalytic processes in continuous-flow millireactors.