Regulation of Microenvironments of Hydrogen-Bonded Organic Frameworks for Enhanced Enzyme Activity of Phosphotriesterase

The microenvironment of the porous channels in enzyme immobilization carriers critically determines the catalytic performance of immobilized enzymes. In this study, we systematically tuned the hydrophobicity/hydrophilicity of the channel microenvironment of hydrogen-bonded organic frameworks (HOFs) by introducing four different substituents (-CH₃, -Cl, -F, -NH₂) at the 2-position of the phenyl ring of the HOF-101 monomer. These HOF-101 derivatives, which are isostructural to the parent HOF-101, were used to immobilize phosphotriesterase (PTE). The enzyme loading efficiencies ranged from 64.7% to 70.7%, indicating that the substituents had little effect on PTE binding, which primarily relies on carboxyl-residue interactions. Kinetic studies revealed that the hydrophilic -NH₂-functionalized HOF-101 (PTE@HOF-101-NH₂) exhibited the highest catalytic efficiency (1.43 × 10⁸ M⁻¹ s⁻¹), 2.27 times that of free PTE, while the hydrophobic -CH₃ analogue showed reduced activity. Notably, PTE@HOF-101-F demonstrated superior acid resistance (70% relative activity at pH 2) and long-term thermal stability (70% activity retention after 6 h at 70 °C), outperforming other derivatives. In contrast, PTE@HOF-101-NH₂ showed the highest activity under mild conditions but suffered from framework dissolution under prolonged harsh treatments. This work demonstrates that fine-tuning the HOF channel microenvironment is an effective strategy to enhance enzyme activity and stability, providing a platform for designing advanced immobilized enzyme systems.