Abstract:

Enzymes are highly efficient biological catalysts, yet their instability outside native environments limits their practical implementation in industrial and technological applications. As such, immobilization within porous materials has emerged as a promising strategy to enhance enzyme stability, recyclability, and catalytic performance. Specifically, among these materials, metal–organic frameworks (MOFs) provide a uniquely tunable platform due to their controllable porosity, crystallinity, and chemical functionality. Despite growing interest in enzyme@MOF (E@MOF) systems, fundamental structure–function relationships governing enzyme encapsulation, crystallization pathways, and spatial distribution remain poorly understood.

This dissertation investigates the formation mechanisms and internal architecture of enzyme-encapsulated MOFs through systematic control of synthetic parameters and the application of advanced cryogenic electron microscopy techniques. First, the role of ligand deprotonation in Metal Azolate Framework-7 (MAF-7) crystallization was established by varying ammonium hydroxide (NH₄OH) concentration and ligand-to-metal ratios. Structural characterization studies using powder X-ray diffraction (PXRD), electron microscopy, and infrared (IR) spectroscopy revealed a critical threshold concentration of deprotonated ligand required for crystalline framework formation, demonstrating that effective ligand availability—rather than total ligand concentration—governs nucleation and growth.

Building upon these findings, enzyme encapsulation within GOx@MAF-7 was examined to determine how base modulation influences enzyme structure, activity, and crystallization pathways. Time-resolved cryogenic transmission electron microscopy (cryo-TEM), combined with enzymatic assays, revealed that NH₄OH concentration modulates nucleation behavior, crystal morphology, and enzyme folding, with higher modulator concentrations leading to partial denaturation and reduced catalytic performance. These results establish modulator chemistry as a key parameter for tuning structure–function relationships in biomimetic mineralization.

Finally, cryogenic scanning transmission electron microscopy–electron energy loss spectroscopy (cryo-STEM-EELS), coupled with multivariate statistical analysis, was employed to directly resolve enzyme spatial distribution within Urease@ZIF-8 composites. This label-free spectroscopic approach enabled nanoscale chemical mapping, revealing preferential enzyme localization near crystal interfaces and distinct bonding environments associated with framework incorporation.

Collectively, this work establishes direct correlations between synthetic conditions, crystallization mechanisms, and enzyme spatial organization in MOF systems, providing a mechanistic foundation for the rational design of biohybrid materials with enhanced catalytic performance and stability.