Abstract
Most cellulose degrading enzymes are composed of modular structures. In the case of fungi, cellulases are formed by a catalytic domain (CD) and by a cellulose-binding domain (CBD), interconnected through a highly glycosylated protein linker. These enzymes are used for
paper production, either to allow a more efficient refinement or to improve the paper sheet
production.
This work aimed at implementing a procedure to produce significant amounts of purified
CDBs. The purified CBDs coating of the fibers surface was assessed by a method based on
fluorescence microscopy. Finally, the application of CBDs in paper production was evaluated.
The CBDs production was achieved by proteolysis, with papain, of the commercial
preparation Celluclast® (cellulases from Trichoderma reesei). Afterwards, the CBDs were
separated by ultrafiltration and further purified by ion exchange chromatography. The obtained
CBDs is part of cellobiohydrolase I, as demonstrated by N-terminal peptide sequencing and
MALDI-TOF. The isolated proteins kept the highly glycosylated peptide linker, as verified by
sugar analysis, that demonstrated the existence of 30% (w/w) of carbohydrates, most of which
mannose (85%). It was shown that these CBDs, as expected, have a high cellulose affinity,
desorbing very slowly. Using CBDs conjugated with fluorescein isothiocyanate (FITC), it was
verified that the adsorption is not uniform, being higher on the fibre extremities and defects, due
either to a higher specific surface area (amorphous regions) or to a higher affinity of CBDs to
these regions.
To quantify the CBDs coating of the fibres’ surface, a MATLAB program was
developed. This program allows for the correlation between the fluorescence signal intensity
produced by the adsorbed CBF-FITC, and its surface concentration. The FITC photobleaching
was verified to be only significant for CBD-FITC in solution, being negligible for the protein
adsorbed on cellulose. The adsorption of CBDs to cellulose films (made from cellulose acetate),
in saturation concentrations, resulted in the equivalent to 1.6 to 2 layer of protein. In the case of
cellulose fibres (Whatman CF11), this value increased to the equivalent of roughly 4 layers.
These high values are due to the protein penetration into the fibers, as was demonstrated by
confocal microscopy and immunolabelling with colloidal gold and transmision electron
microscopy, but also to the increased surface irregularities of Whatman CF11 that would
increase the surface area. The CBD adsorption onto primary and secondary fibres implied a reduction of the
Schopper-Riegler index (increase in water flow between the fibres) and an increase of both the
water retention value (WRV) and air permeability of the handsheets. The strength properties did
not suffer a significant variation with adsorbed CBDs, except for the non-refined virgin fibres. In
this case, a slight improvement was observed, probably due to the lower surface area. The effect
of CBDs on the surface properties was analysed by measuring the ZETA-potencial and contact angles. The adsorption of CBDs chemically conjugated to lysozyme (protein with a positive charge) did not significantly change the papersheet properties. The ZETA-potential was only significantly altered in the case of low surface area fibres (non-refined virgin fibres), as noticed by the reduction of the fibres negative charge. As expected, the variation was more significant with lysozyme conjugated CBD. This higher variation was also confirmed by contact angles measurement. The presence of conjugates made the cellulose film hydrophobic, due to the reduction of the negative polar component of the fibres.
Publication Type: PhD Theses