Self-assembly of mixed-linkage glucan hydrogels formed following EG16 digestion

Mixed-linkage glucans are major components of grassy cell-walls and cereal endosperm. Recently identified plant endo-β-glucanase from the EG16 family cleaves MLGs with strong specificity towards regions with at least four sequential β(1,4)-linked glucose residues. This activity yields a low molecular-weight MLG with a repeating structure of β(1,3)-linked cellotriose that gels rapidly at concentrations as low as 1.0 % w/v. To understand the gelation mechanism, we investigated the structure and behavior using rheology, microscopy, X-ray scattering, and molecular dynamics simulations. Upon digestion, the material's rheological behavior changes from typical polymeric material to a fibrillar network behavior seen for e.g. cellulose nanofibrils. Scanning electron microscopy and confocal microscopy verifies these changes in micro- and nanostructure. Small-angle X-ray scattering shows in-solution self-assembly of MLG through ~10 nm elemental structures. Wide-angle X-ray scattering data indicate that the polymer association is similar to cellulose II, with dominant scattering at d-spacing of 0.43 nm. Simulations of two interacting glucan chains show that β(1,3)-linkages prevent the formation of tight helices that form between β(1,4)-linked d-glucan chains, leading to weaker interactions and less ordered inter-chain assembly. Overall, these data indicate that digestion drives gelation not by enhancement of interactions driving self-assembly, but by elimination of unproductive interactions hindering self-assembly.