ACS Appl Mater Interfaces. 2026 Sep 14. doi: 10.1021/acsami.6c14068. Online ahead of print.

ABSTRACT

Controlling the conformation and orientation of enzymes at solid interfaces is a central challenge in the design of functional biohybrid materials, and the outcome depends sensitively on the chemistry and geometry of the enzyme-support interface. Here, a β-ketoester-functionalized keto-enamine covalent organic framework (COF) is developed as a structurally defined support for arginine-directed immobilization of unspecific peroxygenase (UPO). The crystalline periodicity and post-synthetic addressability of the COF lattice enable precise placement of anchoring groups at the enzyme-support interface. The grafted β-ketoester groups react with surface arginine residues under mild aqueous conditions, forming a robust covalent linkage without external activating agents or toxic cross-linkers. Compared with physical adsorption and lysine-directed epoxy coupling, the arginine-directed strategy offers higher enzyme loading, reduced leaching, and improved reusability, while raising the catalytic efficiency (kcat/KM) toward ABTS oxidation 1.8-fold. Spectroscopic and molecular dynamics analyses indicate that arginine-directed anchoring rigidifies the enzyme and biases the heme channel toward a more open average geometry, offering a structural rationale for the enhanced turnover. The immobilized UPO also shows improved productivity in representative hydroxylation, sulfoxidation, and halogenation reactions. These results establish the identity of the anchoring residue as a tunable interfacial design parameter for COF-supported biocatalytic materials.

PMID:42728243 | DOI:10.1021/acsami.6c14068