Microfluidics-based preparation and in situ immobilization of nanoscale cross-linked enzyme aggregates for continuous transamination

A new microfluidics-based method for the generation and further continuous use of amine transaminase cross-linked enzyme aggregates (ATA-CLEAs) has been developed. The precipitation and cross-linking are carried out in separate but interconnected parts, which allows optimization of each step and thus better final quality of the preparation. After optimizing the acetone and glutaraldehyde concentrations for precipitation and crosslinking, respectively, ATA-CLEAs prepared in a microfluidic device exhibited 87.1% recovered activities, which is 2.4-fold higher than the values previously reported for ATA-CLEAs. The prepared ATA-CLEAs with an average particle radius of 37.13 ± 0.38 nm showed also higher activity than the free enzyme at 70°C. Further integration of a membrane microreactor in the system enabled in situ ATA-CLEA immobilization with 100% yield and up to 68.4% efficiency. The immobilization efficiency and operational stability of the membrane reactor with ATA-CLEAs were more than 20% higher compared to the non-aggregated enzyme