Surface Modification of Cellulose Acetate with Cutinase and ...

Surface Modification of Cellulose Acetate with Cutinase and Cutinase Fused to Carbohydrate-binding Modules
3.2. Progress curves for the modification of cellulose di- and triacetate fabrics and
protein adsorption
Samples of CDA and CTA fabric (1% w/v) were treated with 50 U mL -1 of cutinase for
different periods of incubation. The action of cutinase was evaluated by indirectly
measuring the hydroxyl groups formed at the fibres. Since the cellulose acetates used in
this work were insoluble, the enzyme adsorption to the substrate was a prerequisite for
the formation of the enzyme-substrate complex. The protein adsorption was indirectly
calculated by the decrease in total protein remaining in the treatment solution. For CDA
(figure. 3A), an equilibrium level of relative protein adsorption of 45% (3 mg g -1 of
protein per fabric weight) was reached. For CTA (figure.3B), the equilibrium level of
protein adsorption was higher, with 57% of relative protein adsorption (3.5 mg g -1 ). The
hydrophobic character of the substrate should not be a problem for cutinase adsorption
since this enzyme is a lipolytic enzyme and its natural substrate, cutin, is hydrophobic
(Egmond and Vlieg, 2000; Mannesse et al., 1995; Kolattukudy, 2004).
The formation of hydroxyl groups at the fibre surface was evaluated by staining the
fabric with a cotton reactive dye. The basic principle is the specific reaction between a
vinylsulphonic group from a reactive dye, in this case Remazol Brilliant Blue R, and the
hydroxyl group at the fibre surface. The sensitivity is high due to the large molar
absortivities of dye molecules. If the cutinase is able to hydrolyse some of the acetyl
groups at the surface, then more dye can be chemically linked to the fibre, resulting in
an increase in K/S. The staining ‘titration’ methodology was already reported (Silva et
al., 2005; Matamá et al., 2006, 2007; O’Neill et al., 2007) and proved to be a valuable
and a very sensitive qualitative method.
In the case of CDA fibre, the sensitivity is not as good as for the CTA fibre. The dye has
more affinity for diacetate and, as a result, the controls are very coloured while the
triacetate controls are very faint. This is the reason for the observed differences between
the two fibres in the relative increase in the colour strength values (figure 3).
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