Protein cluster formation during enzymatic cross-linking of globular proteins

Abstract

Work on enzymatic cross-linking of globular food proteins has mainly focused on food functional effects such as improvements of gelation and enhanced stabilization of emulsions and foams, and on the detailed biochemical characterization of the cross-linking chemistry. What is still lacking is a physical characterization of cluster formation and gelation, as has been done for example, for cluster formation and gelation during heat-induced protein aggregation. Here we present preliminary results along these lines. We propose that enzymatic cross-linking of apo-alpha-lactalbumin is a good model system for studying the problem of cluster formation and gelation during enzymatic cross-linking of globular proteins. We present initial results on cluster sizes produced when crosslinking dilute solutions of apo-alpha-lactalbumin with a range of cross-linking enzymes: microbial transglutaminase, horseradish peroxidase, and mushroom tyrosinase. These results are used to highlight similarities and differences between different enzymes, when acting on the same substrate. Next we consider cluster growth and gelation in somewhat more detail for the specific case of cross-linking by horseradish peroxidase, under the periodic addition of H2O2. Upon increasing the initial concentration of apo-alpha-lactalbumin, at a fixed enzyme-to-substrate ratio and fixed reaction time, the size of the clusters at the end of the reaction increases rapidly, and above a critical concentration, gelation occurs. For the conditions that we have used, gelation occurred at very low initial apo-alpha-lactalbumin concentrations of 34% (w/v), indicating a very dilute cross-linked protein network, with a low average number of cross-links per protein. It is found that reactive protein monomers are first rapidly (1-2 h) incorporated into small covalent clusters. This is followed by a much slower phase (up to about 12 h) in which the small clusters are coupled together to form much larger covalent protein clusters. Consistent with this two-step mechanism, atomic force microscopy shows that the covalent protein clusters are very heterogeneous and seem to consist of smaller subclusters.

Cross-linking oppositely charged oil-in-water emulsions to enhance heteroaggregate stability

“…By contrast, the heteroaggregates formed with Quillaja saponins/fish gelatin unexpectedly remained unaffected from the laccase treatment (Figure 2). Supporting our results, Zeeb et al [20] were unable to observe UV/Vis and rheological alterations of laccase-treated fish gelatin solutions, although other disordered coil-like food proteins, such as gelatins, were described in literature as excellent substrates for cross-linking enzymes [21].…”

Assessing the Potential of Folded Globular Polyproteins As Hydrogel Building Blocks

“…Biomolecules provide an almost limitless pool of evolutionary-optimized materials that can be exploited or repurposed to engineer materials with highly specialized functionalities. , Such materials include hydrogels: three-dimensional macroscopic networks swollen by large volumes of water, in some special cases up to 1000× its dry mass . Protein hydrogels use polypeptide chains as the hydrophilic network in order to exploit their intrinsic properties, ,− and they have found applications in tissue engineering, such as vascular grafts and neural tissue regeneration, as well as scaffolds for controlling cell behavior. − In addition, stimulus-responsive protein hydrogels have been explored as ligand-triggered actuators for biosensors and for controlled release for drug delivery. ,,− However, as most protein-based hydrogels are obtained from unstructured peptides or through aggregation of unfolded globular proteins, − the full spectrum of protein function (e.g., catalysis, signaling, and ligand binding) has not yet been exploited. A recent novel approach, that not only obviates these limitations but also harnesses their distinct functional properties, is to build hydrogels from tandem arrayed, folded globular proteins with known mechanical properties. ,, The mechanical properties of the native state of single, monodisperse proteins can be obtained by single molecule atomic force spectroscopy using the atomic force microscope (AFM) − or optical tweezers , as sensitive force transducers.…”

Multivalent-Ion-Activated Protein Adsorption Reflecting Bulk Reentrant Behavior

“…On the one hand, the use of co-solvents such as glycerol to stabilize a protein solution [14] can help to avoid protein aggregation and cluster formation. On the other hand, enzymatic crosslinking [15] or the use of trivalent ions such as yttrium cations can be employed to trigger bridge formation between globular proteins, which can lead to cluster formation, reentrant condensation and liquid-liquid phase separation (Fig. 1) [16,17].…”