Physical and chemical attachment of pectins to substrates : methods, characterisation and application : thesis presented by Abdenor Fellah for the degree of Doctor of Philosophy, Massey University, New Zealand & Fonterra, New Zealand
The plant cell wall is a complex biological matrix in which pectic polysaccharides play an
instrumental role in regulating mechanical properties. Nanomechanical studies of single chains hold
the promise of enabling the comprehension of fundamental aspects concerning the structural,
mechanical and binding properties of pectin at an unprecedented level of molecular detail, using
measured single polysaccharide force-extension behavior as a signature. However, before such
promise can be fulfilled, a better understanding of the attachment of the polymer under study to
the substrates between which it is stretched is required.
Herein, chemoselective methodologies have been developed to covalently couple one end of a
pectin chain onto a solid support. Prior to immobilization, pectin fine structure was investigated
using accurate and non-invasive infrared spectroscopy. Comparison of experimental results with the
predictions of quantum chemical calculations carried out using density functional theory confirmed
this technique as an effective tool for the characterization of pectin fine structure. Subsequently,
following appropriate functionalization of the support, pectin chains were anchored to polystyrene
beads, specifically through their reducing end. These methods were shown to be efficient using IR
spectroscopy, once more coupled with quantum chemical calculations, with the formation of specific
newly introduced bonds being demonstrated.
Finally, single-molecule force spectroscopy was used to stretch single pectin molecules
covalently bonded to substrates using the previously described method applied to glass surfaces.
Compared to physisorption, which was also extensively studied, tethering the pectin non-reducing
end appeared to increase the average stretch length and improved significantly the probability of
stretching a single chain to high forces.