Phase-Separated Multienzyme Condensates for Efficient Synthesis of Imines from Carboxylic Acids with Enhanced Dual-Cofactor Recycling

Enzyme catalysis represents a promising approach for sustainable chemical synthesis, yet its industrial applications face limitations due to the inefficient regeneration and high cost of essential cofactors, such as adenosine-5′-triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). While natural metabolic systems efficiently recycle cofactors through spatially organized enzymes, replicating this efficiency in vitro remains challenging. Here, we prepare a five-enzyme condensate system using liquid–liquid phase separation (LLPS) mediated by intrinsically disordered proteins (IDPs). By colocalizing a carboxylic acid reductase from Norcadia iowensis ( Ni CAR) with a reductive aminase from Aspergillus oryzae ( Asp RedAm) and three cofactor-regenerating enzymes, we generated a phase-separated catalytic condensate that enhanced ATP and NADPH recycling efficiency by 4.7-fold and 1.9-fold relative to free enzymes, respectively. Catalytic performance was correlated with the extent of phase separation, as confirmed by fluorescence microscopy, which revealed clear enrichment of ATP and NADPH within the condensates. This proximity effect enabled efficient cofactor turnover in the one-step reaction, achieving substrate conversion above 90% within 6 h and enhancing the space–time yield (STY) of the chiral imines 1.6-fold, with only one-fifth of the standard cofactor load. This approach creates a scalable and economic tool for performing multienzyme cascade reactions in vitro that are driven by the efficient recycling of multiple cofactors.