Surface Modification of Cellulose Acetate with Cutinase and ...

Subchapter 2.5
identified (Samios, 1997). The deacetylation efficiency of carbohydrate esterases
decreases with the increase in the degree of substitution of cellulose acetate and
consequently its hydrophobicity.
In the work here reported, the hydrolysis of acetate surface groups of cellulose diacetate
(CDA) and cellulose triacetate (CTA) fabrics was investigated using Fusarium solani
pisi cutinase (E.C. 3.1.1.74). It is an extracellular enzyme able to degrade cutin, the
lipid-polyester natural coating of plants, thus conferring phytopathogenicity to the
fungus from which it originates. This enzyme is a small ellipsoid protein (~22 KDa,
45x30x30 Å) that belongs to the class of serine esterases and to the superfamily of α/βhydrolases
(Longhi and Cambillau, 1999). The F. solani pisi cutinase also belongs to
the family 5 of carbohydrate esterases (www.cazy.org/fam/CE5.html), sharing a very
similar 3D-structure with other two members with known structure: the acetylxylan
esterase (E.C. 3.1.1.72) from Trichoderma reesei and the acetylxylan esterase II from
Penicillium purpurogenum (Hakulinen et al., 2000; Ghosh et al., 2001). Although they
present very similar overall structures, the conformation of the active site is different,
reflecting the lipid nature of the cutinase substrates (Ghosh et al., 2001). The preference
for hydrophobic substrates, as well as the versatility in respect to soluble, insoluble and
emulsified substrates makes cutinase an attractive esterase for highly substituted
cellulose acetates.
The enzymatic modification of highly substituted cellulose acetate fibres is a
heterogeneous process. An attempt was made to increase cutinase efficiency towards
this substrate by mimicking other carbohydrate-active enzymes with modular nature.
Two different carbohydrate-binding modules (CBMs) were fused to the C-terminal of
cutinase. The CBMs act synergistically with the catalytic domains by increasing the
effective enzyme concentration at the substrate surface and, for some CBMs, by
physical disruption (Linder and Teeri, 1997; Boraston et al., 2005). Two types of CBMs
were chosen on the basis of ligand affinity, since the two cellulose acetate fibres used in
this work are structurally different from cellulose (the native ligand) and different
between themselves, presenting two different overall crystallinities. Type A, the CBM
of Cellobiohydrolase I (CBHI) from T. reesei belongs to the family CBM1, has
preference for crystalline or microcrystalline regions of cellulose while type B, the
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