New insight into enzymatic cross-linking of globular proteins: from nanostructure to functionality

<strong>ABSTRACT</strong> In last two decades, enzymatic cross-linking of proteins has a growing interest in food technology for better tailoring protein functionality. However, the relation between physical and functional properties of enzymatically cross-linked proteins has been hardly addressed so far. The aim of this thesis was to elucidate the effect of enzymatic protein cross- linking on the physical and functional properties of protein nanoparticles at multiple lengthscale. In the first part of this thesis, as a model system, the enzymatic cross-linking of globular whey protein apo-&alpha;-lactalbumin (&alpha;-LA) by horseradish peroxidase (HRP) was discussed in details. In comparison with HRP, in the second part of the thesis, we also addressed to what extent both laccase (LC) (from trametes versicolor) and tyrosinase (TYR) (from agaricus bisporus) differ in catalyzing oxidative cross-linking of &alpha;-LA. Both HRP and LC were capable of creating self-similar large &alpha;-LA nanoparticles that have an open architecture at similar lengthscales whereas TYR led to the formation of &alpha;-LA oligomers only. All HRP-, LC-nanoparticles and TYR-oligomers exhibited a high extent of secondary structure content preserved whereas their almost all tertiary structure was lost upon enzymatic cross-linking. HRP-catalyzed cross-linking of &alpha;-LA resulted in more hydrophilic nanoparticles than LC-cross-linked &alpha;-LA nanoparticles. Whereas both HRP- and LC-nanoparticles exhibited very high colloidal and thermal stability against protein aggregation at pH 5.8 and 7.0, HRP- nanoparticles were more stable than LC-nanoparticles upon heating and in the presence of dithiothreitol (DTT). This suggests that, unlike HRP- nanoparticles, not only the dityrosine bonds but also disulfide cross-linking stabilizes LC-nanoparticles. Dilute dispersions of HRP-nanoparticles exhibited a high viscosity and a hydrophilic nature. As these dispersions were concentrated, they jammed above their critical overlapping concentration and thus created physical transparent protein hydrogels at relatively low protein concentration (4% w/v). These properties of HRP- nanoparticle dispersions offer high thickening properties that are comparable with polysaccharide in food applications as protein-based thickeners