Cofactor dynamics and diffusional limitations in packed bed flow bioreactors with multi-immobilized enzymes: Ketoreductase and glucose dehydrogenase

• Mechanistic model developed for multi-enzyme packed bed reactor • Cofactor imbalance creates ketone diffusional limitation in the reactor • Excess glucose increases conversion but reduces reactor efficiency • Metrics such as EUM and effectiveness factor reveal hidden inefficiencies • Reactor performance must assess inefficiencies, not conversion alone Asymmetric ketone reduction using immobilized ketoreductase (KRED) coupled with glucose dehydrogenase (GDH) relies on balanced cofactor cycling to sustain efficient biocatalysis. Here, a reaction–diffusion modeling framework is developed to mechanistically decouple the origin and spatial manifestation of cofactor imbalance in a packed-bed bioreactor operated at low ketone concentration (3 mM) to establish baseline behavior. The analysis demonstrates that cofactor imbalance originates from intrinsic kinetic mismatch between GDH and KRED, while diffusion governs its spatial amplification rather than serving as the primary cause. Under excess glucose conditions, rapid NADP⁺ depletion and intraparticle NADPH enrichment generate front-loaded reaction behavior and progressive intraparticle underutilization, reflected by axial variation in the effectiveness factor. Sensitivity analysis confirms that imbalance magnitude is controlled by GDH kinetic parameters, whereas transport modifications alter its severity but not its origin. The reactor exhibits a spatial transition in effectiveness factor from cofactor-controlled behavior near the inlet to substrate-controlled downstream. At elevated ketone feed concentrations (6–9 mM), kinetic–transport coupling intensifies inlet underutilization, while downstream kinetic saturation partially masks diffusional limitations. These results establish a mechanistic framework linking kinetic mismatch and transport effects, demonstrating that conversion alone is insufficient to assess reactor performance and that cofactor dynamics must be explicitly considered in the design of multi-enzyme packed-bed systems.