20.500.12556/RUL-174360 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. amine transaminase enzyme immobilization cross-linked enzyme aggregates membrane microreactor flow biocatalysis amin transaminaza imobilizacija encimov zamreženi encimski agregati membranski mikroreaktor pretočna biokataliza true false true Angleški jezik Slovenski jezik Članek v reviji 2025-10-01 14:01:02 2025-10-01 14:01:04 2026-07-01 03:59:38 0000-00-00 00:00:00 2025 0 0 12 str. Vol. 523, art. 168865 1 Nov. 2025 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 66.098 1385-8947 10.1016/j.cej.2025.168865 251033859 1-s2.0-S1385894725097074-main.pdf 1-s2.0-S1385894725097074-main.pdf 1 639577448B215D206DC8747CAB7C3740 74a0ef15813f803a81aafa8847e9bb2ad3043d32784103629c324e12fd38da15 9159fdf3-7450-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236617 https://www.sciencedirect.com/science/article/pii/S1385894725097074 1 1a664c96-9ebe-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=219155 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0