The interactions of xyloglucans with cellulose: a study using solid-state 13C [i.e. Carbon-13] NMR spectroscopy

Abstract

In this thesis, I describe experiments designed to investigate, using solid-state l3C NMR spectroscopy, the interactions of xyloglucan (XG) and cellulose in cell walls and in model composites of XG and cellulose produced by the bacterium Acetobacter xylinus. I aimed to identify XG adsorbed on to cellulose and in cross-links between adjacent cellulose microfibrils by detecting differences in XG mobility. Wide-angle X-ray scattering (WAXS) diffractometry was also done to characterise further the cellulose in these samples. Cell walls isolated from mung bean (Vigna radiate L.) hypocotyls were examined by solid-state 13C-NMR spectroscopy. To improve the signal-to-noise ratios in these studies, Glc labelled at either C-l or C-4 with l3C-isotope was incorporated into the cell-wall polysaccharides of the mung bean hypocotyls. Using cell walls from seedlings labelled with D-[1-13C]glucose, and by exploiting the differences in rotating-frame and spin-spin proton relaxation, a small signal was detected that I assigned to Xy1 of XGs with rigid glucan backbones. After labelling seedlings with D-[4-l3C]glucose, and using a novel combination of spin-echo spectroscopy with proton spin relaxation-editing (PSRE), signals were detected that had l3C-spin relaxations and chemical shifts which were assigned to partly-rigid XGs surrounded by mobile non-cellulosic polysaccharides. Although quantification of these two mobility types of XG was difficult, the results indicated that the partly-rigid XGs were predominant in the cell walls. The results lend support to postulated new cell-wall models in which only a small proportion of the total surface area of the cellulose microfibrils has XG adsorbed on to it. In my new models, the partly-rigid XGs form cross-links between adjacent cellulose microfibrils and/or between cellulose microfibrils and other non-cellulosic polysaccharides, such as pectic polysaccharides. The structure and the cross-sectional dimensions of the cellulose crystallites in the cell walls from mung bean hypocotyls were examined using cross-polarization/magic angle spinning (CP/MAS) NMR and WAXS diffractometry. CP/MAS NMR indicated that the cellulose consisted of approximately similar proportions of cellulose Iα and cellulose Iβ. The cross-sectional dimension of the cellulose was determined from a WAXS diffractogram to be 2.6 nm. This dimension is consistent with a model with 18 and/or 22 cellulose chains. The cross-sectional dimension estimated from CP/MAS spectra was also consistent with a model in which the cellulose crystallites have 22 cellulose chains. A series of model composites were produced using A. xylinus cellulose and either tamarind XG or XG extracted from the walls of tobacco cell-suspension cultures labelled with D-[1-13C]glucose. These composites were examined by solid-state l3C NMR and WAXS using experiments similar to those used for the cell walls of mung bean. Although XG was associated with all of the model composites, signals were not detected, using solid-state l3C NMR in combination with PSRE editing, that could be assigned to XG adsorbed on to the surface of the bacterial cellulose. However, both the tamarind XG and the XG from I3C-labelled tobacco cell walls showed proton and l3C-relaxation NMR characteristics similar to those shown by the XGs in the cell walls from mung bean hypocotyls and which were assigned to XG forming cross-links between adjacent cellulose microfibrils. By using spin echo NMR in combination with CP/MAS PSRE and with single-pulse excitation/magic-angle spinning (SPE/MAS) NMR, I found that the XG detected by CP/MAS had the same mobility as the XG detected by SPE/MAS, indicating that XG responding to CP/MAS NMR also responds to SPE/MAS. Both WAXS and solid-state 13C NMR results indicated that the cross-sectional dimensions of the crystallites of bacterial cellulose were considerably larger than those of mung bean cellulose. Therefore, I propose that the proportion of cellulose with adsorbed XG in the composites containing bacterial cellulose was below the limits of detection by solid-state 13C NMR. CP/MAS NMR of the model composites indicated that cellulose Iβ was the dominant allomorph in the crystallite interior of a composite in which bacterial cellulose was produced in culture medium containing tamarind XG and then heated. Cellulose Iα was the dominant allomorph in the cellulose crystallites of a composite in which cellulose was produced in medium containing XG but was not heated. WAXS diffractograms and CP/MAS NMR spectra indicated that in both of these composites, the cross-section dimensions of the cellulose crystallite was reduced compared with bacterial cellulose produced in medium with no XG, unheated or heated. This suggests that although the cellulose crystallites were smaller, that is disrupted by the addition of XG to the medium, it was not until the cellulose was heated that cellulose Iβ became the dominant allomorph. Moreover, these results are consistent with XG interacting with the surface molecules of cellulose, despite XGs with rigid backbones not being detected by the techniques used.