Confined laccase-mediator synergy within a hydrogen-bonded organic framework nanoreactor

The practical application of laccase in environmental biocatalysis is fundamentally limited by its fragile protein structure and restricted redox potential toward non-phenolic substrates. While laccase-mediator systems (LMS) electrochemically broaden this substrate scope, the homogeneous reaction fails to provide the spatial confinement necessary for durable mediator retention and enzyme protection. Herein, we conceptualize a nanoconfined bio-abiotic nanoreactor via the synchronous, de novo encapsulation of laccase (LAC) and the mediator 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) the crystalline nanochannels of a hydrogen-bonded organic framework, HOF-101, a metal-free platform that self-assembles under mild aqueous conditions without metal ions. This deliberate co-localization enforces a proximity synergy that short-circuits the electron transfer pathway. The resulting LAC+ABTS@HOF-101 composite exhibits a catalytic efficiency (k_cat/K_m) of 78.26 s^-1 mM^-1, a 2.7-fold enhancement over free laccase. The HOF nanochannels function as a rigid exoskeleton, endowing the enzyme with profound conformational stabilization: 92% activity is retained at 60 °C, and a high 176% relative activity is maintained under prolonged trypsin challenge, demonstrating strong resistance to proteolytic degradation. In the decolorization of crystal violet, a model recalcitrant dye, the nanoreactor achieves near-quantitative 96% removal. Crucially, the physically constrained ABTS is durably retained and regenerated in situ within the nanochannels, enabling 67% decolorization efficiency after five consecutive cycles, while a non-confined control system catastrophically fails after a single run. This work establishes a paradigm for engineering multi-component enzymatic systems through deliberate spatial organization, opening new avenues for robust biocatalytic materials in environmental remediation.