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.
Wu et al. (Thu,) studied this question.