Engineering and immobilization of imine reductase enable chemoenzymatic synthesis of SLC6A19 inhibitor JNT-517.

Enzymes represent renewable biological resources, and their employment as industrial biocatalysts not only enhances production efficiency but also mitigates environmental pollution associated with excessive organic solvent usage. (R)-N-Cyclopropylpiperidin-3-amine serves as a crucial pharmaceutical intermediate, traditionally produced via chiral resolution. Imine reductases (IREDs) present an atom-efficient alternative by directly catalyzing the reductive amination of prochiral ketones with amines to yield chiral alkylated amines. Through site-directed mutagenesis of IR-G36-M5, we developed the double mutant M203A/S241L, which demonstrated enhanced enantioselectivity (improving from 87 % to > 99 % ee for the R-configuration) and a 38 % increase in catalytic efficiency. Subsequent immobilization in ZIF-8 further improved the mutant's thermal stability and solvent tolerance. M203A/S241L@ZIF-8 retained 40 % residual activity after 21 days at ambient temperature. The immobilized enzyme system exhibited robust operational stability (> 80 % conversion maintained after 5 reuse cycles) and achieved a high space-time yield of 4.7 g g-1 h-1 in batch operations. This biocatalytic system was successfully integrated into the chemoenzymatic synthesis of SLC6A19 inhibitor JNT-517, where the enzymatically produced (R)-N-cyclopropylpiperidin-3-amine was efficiently converted to JNT-517 in three chemical steps, demonstrating promising potential for industrial-scale applications. Protein engineering and enzyme immobilization technology endow IRED with superior catalytic performance, stability, and reusability, thereby enabling sustainable bioprocessing for industrial applications.