Microfluidics-based generation and immobilization of nanoscale cross-linked enzyme aggregates for continuous transamination

Cross-linked enzyme aggregates (CLEAs) offer a cost-effective and robust strategy for enzyme immobilization, combining high recovered activity with improved operational stability—key traits for continuous biocatalysis. However, conventional batch preparation methods typically yield heterogeneous, micrometer-sized particles with poor reproducibility, mechanical fragility, and limited applicability in continuous reactor systems. In this study, we introduce a novel microfluidic approach for generating stable, uniform nanoscale CLEAs of amine transaminase (ATA-CLEAs). By spatially separating acetone-induced precipitation and glutaraldehyde cross-linking into connected microfluidic sections, we enabled independent optimization of both steps. This system yielded highly uniform ATA-CLEAs (~100 nm diameter) with up to 90.5 % recovered activity—2.5-fold higher than batch-prepared aggregates— while reducing processing time and reagent consumption. Additionally, we demonstrate the first integration of CLEA synthesis with a membrane microreactor, enabling one-step purification and immobilization with 100 % yield. Upon connection to a substrate feed, the system was seamlessly adapted for continuous transamination of ( S )-α-methylbenzylamine with pyruvate, achieving over 68 % immobilization efficiency. The immobilized ATA-CLEAs demonstrated superior operational stability compared to their non-aggregated counterparts, delivering a 45 % higher turnover number over five days of continuous operation. This work establishes a scalable and efficient platform for incorporating CLEAs into continuous biocatalytic processes—an essential advancement in green engineering for the sustainable production of pharmaceuticals and fine chemicals. • Cross-linked amine transaminase enzyme aggregates were generated using microfluidics. • Very uniform particles of below 100 nm in diameter were obtained. • Recovered activity of up to 90.5 % is the highest reported for transaminase aggregates. • Immobilization in a membrane microreactor enabled continuous biotransformation. • Aggregates outperformed the operational stability of membrane-immobilized enzymes.