Immobilized enzymes in continuous biotransformations: advances, challenges, and engineering perspectives for scalable biocatalytic processes

Immobilized enzymes have become central to continuous biotransformations by enabling catalyst recovery, repeated utilization, simplified product separation, and stable long-term reactor operation. This study critically evaluates how immobilization strategies, support architectures, reactor configurations, and reaction media collectively govern enzyme stability, mass transfer, catalytic efficiency, and process productivity in continuous biocatalytic systems. Recent advances in adsorption, covalent immobilization, entrapment, encapsulation, cross-linked enzyme aggregates (CLEAs), membrane reactors, packed-bed reactors, monolithic reactors, and structured flow platforms are systematically assessed with emphasis on industrially relevant performance metrics. The analysis demonstrates that although immobilization substantially enhances operational stability and catalyst lifetime, overall process performance is dictated by the interplay between enzyme orientation, support microstructure, pore diffusion, substrate and product transport, water activity, cofactor availability, product inhibition, enzyme leaching, fouling, and hydrodynamic limitations. Despite significant progress, meaningful comparison across studies remains constrained by inconsistent reporting of key process parameters, including enzyme loading, immobilization efficiency, residence time, space-time yield (STY), turnover number (TON), operational half-life, catalyst productivity, pressure drop, and time-on-stream stability. The study identifies critical knowledge gaps in mechanistic understanding, standardized performance evaluation, and long-duration continuous operation. It proposes an integrated framework that combines rational immobilization design with transport modeling, in situ reaction diagnostics, and cofactor engineering.