Immobilization of His₆-tagged amine transaminases in microreactors using functionalized nonwoven nanofiber membranes

Process intensification is crucial for industrial implementation of biocatalysis and can be achieved by continuous process operation in miniaturized reactors with efficiently immobilized biocatalysts, enabling their long-term use. Due to their extremely large surface-to-volume ratio, nanomaterials are promising supports for enzyme immobilization. In this work, different functionalized nanofibrous nonwoven membranes were embedded in a two-plate microreactor to enable immobilization of hexahistidine (His₆)-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu²⁺ ions gave the best results regarding His₆-tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N-His₆-ATA-wt enzyme. Moreover, an efficient one-step enzyme immobilization process from overproduced enzyme in Escherichia coli cell lysate was developed and yielded enzyme loads up to 1088 U mL^-1. High enzyme loads resulted in up to 80 % yields of acetophenone produced from 40 mM (S)-α-methylbenzylamine in less than 4 min using a continuously operated microreactor. Up to 81 % of the initial activity was maintained in a 5-day continuous microreactor operation with immobilized His₆-tagged ATA constructs. The highest turnover number within the indicated time was 7.23·10⁶, which indicates that this immobilization approach using advanced material and reactor system is highly relevant for industrial implementation.