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

Menegatti, Tadej (author), Lavrič, Žan (author), Hlebanja, Patrik (author), Žnidaršič Plazl, Polona (author)

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.

• Language: English
• Keywords: amine transaminase, enzyme immobilization, cross-linked enzyme aggregates, membrane microreactor, flow biocatalysis
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: 12 str.
• Numbering: Vol. 523, art. 168865
• PID: 20.500.12556/RUL-174360
• UDC: 66.098
• ISSN on article: 1385-8947
• DOI: 10.1016/j.cej.2025.168865
• COBISS.SI-ID: 251033859
• Publication date in RUL: 01.10.2025

Record is a part of a journal

• Title: Chemical engineering journal
• Shortened title: Chem. eng. j.
• Publisher: Elsevier
• ISSN: 1385-8947
• COBISS.SI-ID: 2110998

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: amin transaminaza, imobilizacija encimov, zamreženi encimski agregati, membranski mikroreaktor, pretočna biokataliza