Proximity-enhanced co-immobilized enzyme cascade for efficient bioconversion of nicotine to 3-succinoylpyridine

Tobacco waste, rich in nicotine, is both an environmental burden and a potential feedstock for high-value chemicals. Here, we developed a proximity-enhanced co-immobilized multi-enzyme cascade that efficiently converts the nicotine into a pharmaceutical intermediate 3-succinoylpyridine (SP). The cascade system comprises the nicotine oxidoreductase NicA2 V321 (NicAm), pseudooxynicotine amine oxidase (Pnao), and 3-succinoylsemialdehyde-pyridine dehydrogenase (Sapd) for sequential nicotine conversion, coupled with an aldehyde–ketone reductase (AKR) module for in situ NADP⁺ regeneration. To improve nicotine-to-SP conversion and facilitate multi-enzyme recycling, a SpyCatcher/SpyTag self-assembled cofactor regeneration enzyme scaffold was adopted, in conjunction with AviTag–BirA-mediated biotinylation for cascade enzyme site-specific co-immobilization on streptavidin-coated supports. This proximity-enhanced design promoted efficient cofactor cycling and boosted the nicotine-to-SP conversion to approximately 63%, much higher than the 42.7% achieved by free enzymes. The co-immobilized system also showed improved pH and thermal stability, retaining over 60% of its initial activity after eight reuse cycles. This modular biocatalytic strategy provides a green and promising route for the valorization of nicotine-rich tobacco waste.