Surface Modification of Cellulose Acetate with Cutinase and ...

Subchapter 2.5
spacers was studied by several authors mainly through deletion studies. It was
demonstrated that linker peptides, connecting the catalytic domains of carbohydrate-
active enzymes and the CBMs, are necessary for the synergistic activity between the
two domains (Srisodsuk et al., 1993; Shen et al., 1991). The wild-type linker of CBHI
was included in the fusion protein with the CBM from the same enzyme. A smaller
linker was also used to connect cutinase to the fungal CBM (figure. 1). The initial
purpose was to increase the levels of expression in E. coli of the soluble cutinase fused
to CBMCBHI, by removing from the wild-type linker a sequence of residues that
constitute possible sites for O-glycosylation. Since E. coli does not possess the
machinery necessary for this post-translation eukaryotic modification, removing those
residues could promote correct folding of the fusion protein. The expression levels were
very low for soluble cutinase-wtCBMCBHI and were not significantly improved in the
case of cutinase-sCBMCBHI. The bacterial linker used was the proline-threonine box
(PT)4T(PT)7 present on the CenA from C. fimi (Shen et al., 1991). This type of PT
linker is also naturally glycosylated, but when it is not, the conformations of catalytic
domain and CBM are preserved, since only a partial increase in the linker flexibility
seems to occur (Poon et al., 2007).
In the treatment of CDA and CTA with cutinase and its fusion proteins, it was not
possible to detect acetic acid as previously. For longer treatments, the quantification of
acetic acid was somehow impaired. The reasons could be some volatility,
microbiological contamination (in spite of the sodium azide) and/or different
efficiencies of cutinase from different batches.
Protein quantification after treatment with cutinase-CBMN1 and cutinase-PTboxCBMN1
was unviable due to the turbidity of solutions. This turbidity happened only for the
referred assays, where protein adsorption might be underestimated. The turbidity could
be precipitated protein or due to non-hydrolytic disruption of cellulose acetate fibres, in
particular, of CDA for which this phenomenon was most visible. This mechanical
disruption was already described for cellulose and cotton in the presence of CenA, Cex
and isolated CBMs (Din et al., 1991 and 1994, Cavaco-Paulo et al., 1999). Comparing
the amount of protein adsorbed and relative K/S between chimeric proteins and
cutinase, there was a clear difference between the two cellulose acetates studied (figure.
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