Amine-reactive hydrogels via nucleophilic thiol-yne reaction: A functional scaffold for laccase immobilization and...

In this study, we synthesized a novel six-armed propiolic ester as a crosslinker to prepare PEG-based amine-reactive hydrogels via the thiol-yne 'click' reaction. By adjusting the molar ratios of components, three hydrogels with different compositions were obtained. All those hydrogels contained vinyl sulfide groups at junction points capable of nucleophilic addition, two of the formulations additionally retained residual propiolate functionality for post-modification through the amino-yne click reaction. Characterization of fabricated hydrogels was done by FTIR, TGA, swelling studies, and SEM. Covalent immobilization of Trametes versicolor laccase was achieved through reactions between amine groups of the enzyme and the reactive functionalities of the hydrogels. We observed that similar amounts of enzyme were immobilized on all hydrogel formulations, however there were significant differences in dye decolorization efficiency. The laccase-immobilized hydrogel with the highest density of accessible propiolate groups exhibited the greatest dye decolorization efficiency (89.71%), whereas the formulation lacking accessible propiolate groups showed only 25.59% decolorization. After subtracting the adsorption contribution of the bare hydrogels, the net enzymatic degradation efficiency increased from 17.03% in the hydrogel lacking accessible propiolate groups to 83.18% in the hydrogel containing the highest density of accessible propiolate functionalities. This difference was attributed to the increased accessibility of the active site of the enzyme and enhanced substrate diffusion due to lower crosslinking density. The laccase-hydrogel conjugate, showing the highest enzymatic activity displayed also good storage stability and reusability. These results demonstrate the potential of propiolic ester-based hydrogels as versatile platforms for enzyme immobilization and biocatalytic applications.