An amino acid-fortified in situ encapsulation strategy for constructing highly active enzyme@HOF biocomposites

Abstract

Carboxylic acid dimerization-directed hydrogen-bonded organic frameworks (HOFs) have emerged as promising protective scaffolds for enzyme immobilization via a facile in situ encapsulation process. However, apart from the dominating hydrogen bonds, the construction of stable HOF scaffold involves additional interactions, e.g., π-π stacking interactions, stemming from an extended π-conjugated linker structure. This inevitably introduces organic solvents for dissolution of the hydrophobic linkers, leading to adverse effects on the environment, human health and enzyme activity. Therefore, in this work, a benign amino acid-fortified in situ HOF encapsulation strategy is presented under truly aqueous, biocompatible conditions. Attributed to the proton sponge effect of basic amino acids for deprotonating the carboxyl (-COOH) groups of HOF linkers, water-soluble linker-amino acid complexes are formed through electrostatic interactions between the anionic linkers and cationic amino acids. The extendibility of this strategy has been verified across diverse carboxylate HOF linkers. In addition, after a simple controlled acidification, the protonated linkers are recovered to neutral state which promote their crystallization onto the enzyme surfaces to afford enzyme@HOF biocomposites. In the case of N,N-dimethylformamide (DMF)-sensitive enzyme (for instance, horseradish peroxidase), this His-fortified in situ HOF encapsulation endows the resulting HOF biocomposites with 2.5 to 333.3-fold enhancements in bioactivity compared to the conventional DMF-mediated HOF recrystallization method. Furthermore, the recovery of -COOH groups significantly facilitates the enzyme loading content in the biocomposites due to the increased available hydrogen bonding sites while well preserving bioactivity. This work offers a sustainable platform for advanced bio-nano catalytic systems while addressing critical environmental and biocompatibility concerns in enzyme immobilization.