Surface engineering of amine transaminases to control their region-selective immobilization.

The industrial use of enzymes often requires their immobilization to facilitate downstream processing and enable reuse. However, controlling enzyme orientation during immobilization is challenging and typically restricted to the N- and C-terminal regions. In this work, we propose a strategy to immobilize more active and stable amine transaminases (ATAs) by combining protein engineering with immobilization techniques. Our approach involves the structure-guided insertion of histidine clusters (His-clusters) at flexible regions of ATA subunit interfaces, enabling immobilization on cobalt-chelated carriers. By screening multiple ATAs from various microbial sources and testing different His-clusters for each, we identified the most active and stable heterogeneous biocatalysts. Notably, the immobilized H2A variant of Chromobacterium violaceum ATA (CvATA-2HA) exhibited the highest activity per mass of biocatalyst (4 U g). Meanwhile, the H3 variant of Pseudomonas fluorescens ATA (PfATA-H3) showed enhanced thermostability and DMSO resistance, being approximately 2.5 times more stable than its free counterpart. Overall, our findings highlight the impact of enzyme surface engineering on immobilization efficiency. The strategic placement of His-clusters enabled region-directed immobilization, improving both the activity and stability of specific ATA variants.