Hierarchically engineered enzyme-packed bed reactor via 3D scaffold design and zwitterionic microenvironment for sustainable continuous catalysis.

Immobilized enzyme packed bed reactors have contributed significantly to green and sustainable chemistry, yet design strategies at both molecular and system levels are needed. In this study, polylactic acid scaffolds were printed using 3D printing. The microporous scaffold with a high specific surface area was obtained through an etching-activation process. The enzyme sources and polymer microenvironments of site-specific immobilized His-tagged penicillin G acylase were screened. The polysulfobetaine methacrylate microenvironment displayed better relative activities, affinity, storage stability, and thermal and pH tolerance of enzyme compared to the PEG microenvironments. Packed bed reactors were constructed using scaffolds with different pore sizes, and the space-time yields were investigated. The internal flow behavior was studied using flow-field simulation and average residence time distribution. This study not only provides a robust multi-level design strategy for immobilized enzyme packed bed reactors but also presents new protocols for medical wastewater treatment and penicillin production.