Chiral amines production by compartmentalized enzymes in membrane-based flow bio-catalysis

• Nanoporous membranes enabled compartmentalized multi-enzyme catalysis, overcoming incompatibility and inhibition. • Production of highly pure amino acids with high enantiomeric excess in flow catalysis. • Co-factor recycling supports scalable and continuous biocatalytic processes. Multi-enzyme biocatalysis can mimic the synthetic capabilities of natural metabolic pathways but faces significant challenges, including the need for enzyme immobilization and cofactor recycling, separation of incompatible enzymes, and mitigation of product inhibition. Here, these limitations are addressed by compartmentalizing enzymes and cofactors within a nanoporous membrane used in flow mode. Two incompatible enzymes, a protease and a ω-transaminase, were successfully retained within distinct polydopamine/polyethyleneimine layers of the membrane. Pure enantiomers of amino acids were generated from racemic methyl ester precursors via the enzymatic membrane. As the flow rate across the membrane can be precisely controlled, residence time and substrate conversion can be optimized and inhibitory ketone byproducts removed. At optimal conditions, the enantiomeric excess reached 97 ± 2 %. Cofactor retention was achieved by electrostatic trapping of pyridoxal-5′ phosphate within a polyelectrolyte layer of the membrane; a single application of cofactor was effective for 3 cycles of flow catalysis thereby reducing cost. The compartmentalization of enzymes and cofactors within membranes operated in flow mode offers a new pathway for conducting multi-step biocatalysis which is especially suited for the separation of incompatible enzymes and the continuous removal of inhibitors, resulting in a flexible and readily scalable approach.