Anchoring Effect-Induced Conformation Remodeling in Epoxy-Functionalized Covalent Organic Frameworks for Enhanced Enzymatic Efficiency

Covalent organic frameworks (COFs) with tunable structures and versatile chemical functionalities offer great promise for enzyme immobilization. This study explores the "anchoring effect" induced by covalent bonding, which significantly enhances enzymatic efficiency. Glucose oxidase (GOx) was covalently immobilized onto an epoxy-functionalized COF (<i>o</i>COF-TATP) by incorporating epoxy groups into the COF framework. The covalent immobilization approach increased the enzyme loading from 26 mg/g (physical adsorption) to 40 mg/g. Secondary structure analysis revealed that the covalent binding promoted a shift from the α-helix to the β-sheet structure, with a more substantial effect than physical adsorption. Molecular dynamics simulations further confirmed that the anchoring effect enhanced structural rigidity and exposed the enzyme's active site. This led to an 8.14% increase in accessible surface area and a 7.55% rise in hydrogen bonding interactions. These structural improvements resulted in significant catalytic enhancements, with the turnover rate (<i>k</i>_cat) and catalytic efficiency (<i>k</i>_cat/<i>K</i>_M) of GOx ⊂ <i>o</i>COF-TATP reaching 197 and 388% of the free enzyme, respectively. In glucose oxidation assays, the immobilized enzyme exhibited 115% of the product compared to free GOx, maintaining over 95% activity after six reuse cycles and demonstrating superior stability under various conditions. The "anchoring effect" represents a novel mechanism for optimizing enzyme performance and operational stability.