Enzymatically tailored xyloglucan and its performance in in vitro assembled cellulose / xyloglucan composites

Abstract

Xyloglucans are an important class of plant hemicellulosic polysaccharide and fulfil two roles in plants. They are major components of the specialised storage cell walls of some seeds, functioning as energy reserves. They are also present in primary cell walls where they are associated with cellulose in the cellulose/xyloglucan network This network is considered to play a role in the maintenance of structural integrity and the regulation of cell growth. To examine the way in which the structural features of xyloglucan affect the xyloglucan/cellulose interaction an in vitro system modelling the network has been developed (Whitney SEC, Brigham JE, Darke AH, Reid JSG, Gidley MJ, The Plant Journal, 8, 1995). This involves extrusion of cellulose by a Gram negative bacterium Acetobacter xylinus (formerly Acetobacter acetii ssp. xylinum ATCC 53524) into a medium containing xyloglucan resulting in a xyloglucan/cellulose matrix. This thesis examines the interaction between enzymatically tailored tamarind seed xyloglucan and in vitro produced cellulose. Four xyloglucan-specific hydrolases were purified from nasturtium cotyledons. Three of these enzymes, p-galactosidase, nasturtium xyloglucan endoglucanase / endotransglycosylase (NXET), and a-xylosidase, were then used to obtain structurally modified xyloglucans. The performance of these tailored xyloglucans in the Acetobacter model system was then investigated using biochemical techniques, ’^C-NMR, Uniaxial Tensile Testing and Deep-Etch Freeze-Fracture Transmission Electron Microscopy. The p-galactosidase was used to produce xyloglucans with 30% and 60% galactose depletion and characterisation of the resulting composites methods showed that increased removal of galactose from xyloglucan reduced its solubility and caused it to self-aggregate, rendering it unavailable for association with cellulose. Several approaches to the selective removal of xylose were explored. Xylose-depleted xyloglucan was obtained by the concerted action of a-xylosidase and NXET. Characterisation of the resulting cellulose/xyloglucan composites showed very little association with cellulose due to the lowered molecular weight of the tailored xyloglucan.