Pea albumins are the soluble fraction of pea proteins, recovered as a side stream of the isoelectric precipitation. The time-resolved heat-induced structural transitions of the whole fraction of pea albumin (PA) were studied by evaluating the structure of the two main fractions, PA1 and PA2. Synchrotron radiation circular dichroism (SR-CD) spectroscopy showed that the secondary structure signature for PA1 remained unchanged at temperatures up to 85 °C. In contrast, PA2 underwent a change in secondary structure between 45 and 70 °C. These thermal transitions were also confirmed by Nano Differential Scanning Calorimetry (Nano-DSC). Small-angle X-ray scattering (SAXS) in situ experiments were also carried out, and high-resolution structural models of PA1 and PA2, derived from the AlphaFold Protein Structure Database, were used for data analysis. PA1 maintained a consistent local structure with a radius of gyration ∼17 Å across all temperatures, but formed small soluble aggregates above 60 °C, as evidenced by an upturn at low q. PA2 scattering resulted from its dimeric structures and transitioned to unfolded structures after 60 °C. The unfractionated PA scattering showed good agreement with a linear combination of PA1 and PA2 scattering. This was also confirmed by the structural behavior of the purified PA1+PA2 mixture, which closely resembled that of PA2, though the PA2 component remained partially folded even after heating above 70 °C. This study brings new knowledge on the contribution of the two main components of pea albumins in the thermal behavior of this novel extract, with great potential to be used as a functional food ingredient.

Iodine is a well-established antimicrobial agent, but its clinical use is limited by low chemical stability and adverse effects. This work investigates iodine loading into degradable starch microspheres (DSM), elucidating iodine-starch interactions, and structural changes after iodine uptake. Iodine complexation was achieved using a KI/I₂ system, yielding a characteristic purplish-red coloration within the DSM. Swelling and sedimentation analyses showed a slight reduction in DSM density after iodine absorption, without negative impact on hydration capacity. A UV-Vis spectrophotometric method targeting the triiodide band at 352 nm enabled robust quantification of iodine by leveraging equilibrium among iodine species. Sorption isotherms revealed cooperative binding behavior, with iodine uptake increasing nonlinearly in response to the external iodine concentration. Small- and wide-angle X-ray scattering (SWAXS) showed iodine-induced ordering across multiple length scales, including a reduction in the dynamic correlation length and the emergence of characteristic peaks associated with iodine-amylose complex. These results elucidate the physicochemical mechanisms governing iodine incorporation into DSM and highlight the structural consequences of loading, providing a framework for designing starch-based carriers for controlled iodine release.

Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes that bind to and oxidize insoluble carbohydrate substrates such as chitin or cellulose. This class of enzymes has attracted considerable attention due to their ability to convert biomaterials of high abundance into oligosaccharides that can be useful for producing biofuels and bioplastics. However, processes at the interface between solution and insoluble substrates represent a major challenge to biochemical and structural characterization. Here, we investigated the four-domain LPMO from Vibrio cholerae, N-acetyl glucosamine binding protein A (GbpA), to elucidate how it docks onto its insoluble substrate with its two terminal domains. First, we developed a protocol that allowed GbpA and chitin to form a stable complex in suspension, overcoming incompatibilities of the two binding partners with respect to pH. After determining the small-angle neutron scattering (SANS) contrast match point for chitin (47% D2O), we characterized the mesoscale structure of GbpA in complex with chitin by SANS and complemented the results with negative-stain electron microscopy. We found that GbpA binds rapidly to chitin, where it coats the chitin fibers and smooths their surface. In some locations, GbpA binding induces the formation of protein-chitin clumps containing a large number of GbpA molecules. Together, this suggests how the secretion of GbpA efficiently prepares the ground for microcolony formation by the bacteria.

The potential of using proteins as drugs is held back by their low stability in the human body and challenge of delivering them to the site of function. Extensive research is focused on drug delivery systems that can protect, carry, and release proteins in a controlled manner. Of high potential are cross-linked degradable starch microspheres (DSMs), as production of these is low-cost and environmentally friendly, and the products are degradable by the human body. Here, we demonstrate that DSMs can absorb the model protein lysozyme from an aqueous solution. At low amounts of lysozyme, its concentration in starch microspheres strongly exceeds the bulk concentration in water. However, at higher protein contents, the difference between concentrations in the two phases becomes small. This indicates that, at lower lysozyme contents, the absorption is driven by protein-starch interactions, which are counteracted by protein-protein electrostatic repulsion at high concentrations. By applying small-angle X-ray scattering (SAXS) to the DSM-lysozyme system, we show that lysozyme molecules are largely unaltered by the absorption in DSM. In the same process, the starch network is slightly perturbed, as demonstrated by a decrease in the characteristic chain to chain distance. The SAXS data modeling suggests an uneven distribution of the protein within the DSM particles, which can be dependent on the internal DSM structure and on the physical interactions between the components. The results presented here show that lysozyme can be incorporated into degradable starch microspheres without any dependence on electrostatic or specific interactions, suggesting that similar absorption would be possible for pharmaceutical proteins.