Page 294 - Advances in Textile Biotechnology

Enzymatic functionalization of cellulosic fi bres for textiles 275

This technology requires three essential components (Fig. 11.3): (1) high

molar mass XG polysaccharide, (2) chemically modified xyloglucan oligo-
saccharides (XGO-R), and (3) a xyloglucan endo-transglycosylase (XET).
1. Xyloglucan polysaccharide (XG). For reasons of convenience and large-
scale availability (vide supra), tamarind seed XG is the base material of
choice. The oligosaccharide composition of this polysaccharide is well-

established, and the native high molar mass is sufficiently high (Mw >
500 000).

2. Chemically modified xylogluco-oligosaccharides (XGO-R). These
enzyme substrates are readily produced by the digestion of tamarind
XG with microbial xyloglucanases (Gilbert et al., 2008; Pauly et al.,
1999b; York et al., 1993), followed by reductive amination to install an
amino group on the first Glc residue of the XGO (Fig. 11.4). This amino

group provides a uniquely reactive chemical handle, which may be
further functionalized, depending on the application. Reductive amina-
tion is practically convenient because the reaction occurs in aqueous
solution without the need for hydroxyl protecting groups. Chemical
handles may also be installed by anomeric glycosylation (Ibatullin et al.,
2008), lipase-catalyzed esterifi cation (Gustavsson et al., 2005), or via
oxidation with galactose oxidase (Priem et al., 1997). Xyloglucanase
digestion of tamarind XG produces a mixture of the four constituent
oligosaccharides (Figs 11.1 and 11.4) and, although this may be simpli-
fi ed by β-galactosidase digestion and/or chromatographic separation
(Greffe et al., 2005), it is practically more convenient to use the mixture
directly on a large scale.
3. Xyloglucan endo-transglycosylase (XET). This enzyme is responsible
for the recombination of the XGO-R substrates into the XG polysac-
charide. Work in our laboratory has routinely employed a XET,
PttXET16-34 from the hybrid aspen (Populus tremula × tremuloides),
produced recombinantly in the yeast Pichia pastoris (Bollok et al., 2005;
Kallas et al., 2005). Recombinant expression has the distinct advantage
over extraction from native sources that it is highly scalable and indus-
trially widespread (Bollok et al., 2005). PttXET16-34 is biochemically
well-characterized (Kallas et al., 2005), including three-dimensional
enzyme structural analysis (Johansson et al., 2004). Notably, these struc-
tural studies, which include XET-XGO ligand complexes, indicate that
this enzyme has an active site cleft with the potential to tolerate a diver-
sity of sterically bulky XGO-R functional groups.
Admixture of these three components in buffered aqueous solution results
in the XET-catalyzed incorporation of XGO-R into high mass XG, to
yield functionalized XG, XG-R, with a concomitant reduction in polysac-
charide chain length (Fig. 11.3, step a). Subsequent adsorption of XG-R

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