Projects

Principal Investigator

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Team Members - CEB

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Abstract

Enzymes have long been applied in the paper industry, for the modification of both fiber and paper properties. However, several drawbacks result from this utilisation. Hydrolysis of polysaccharides leads to a reduction of both fiber strength and mass. To avoid these side effects, the use of cellulose-binding modules (CBMs) in papermaking has been recently evaluated. CBMs are non-catalytic modules present in several cellulases and hemicellulases that exhibit high affinity for cellulose. These modules are produced by a large number of fungi and bacteria and potentiate the catalytic efficiency of the associated catalytic domains. Previous work conducted in our research group showed that fungal CBMs, heavily glycosylated, modify the properties of textiles and paper pulps. This effect may be assigned, theoretically, either to the modification of interfacial properties or to the disruption of the fibers. Subsequently, we expressed a CBM in the bacterium Escherichia coli and tested it in the treatment of paper fibers. Although both the native fungal CBM obtained by proteolysis and the non-glycosylated recombinant CBM presented high affinity for cellulose, the later did not modify the properties of paper pulps. This may be due to the lack of glycosylation, which may be relevant concerning the modification of the fibres surface properties, since glycosylation is lacking in the bacterial protein. To confirm this hypothesis, the recombinant CBM was chemically conjugated with activated polyethylene glycol (PEG) to mimetize the glycosidic fraction present in fungal CBM which, as PEG, is likely to be highly hydrated. The adsorption of the CBM-PEG conjugate into the fibers probably would modify the paper surface wetability. Indeed, CBM-PEG conjugate improved pulp and paper drainability, a parameter closely related to high energy spending in papermaking, without affecting negatively the physical properties of the papersheets. The results clearly indicated, for the first time, that glycosylation is required for the benefits of fungal CBMs in papermaking, as the recombinant non-glycosylated CBM did not have any effect on fibers. Of course, the functionalization of recombinant CBMs with PEG is not economically attractive. In addition, high quantities of pure functional CBMs are required for their successful industrial application. The development of cheaper alternative strategies to obtain pure glycosylated CBMs is thus desired. This goal may be achieved by the recombinant production and purification of glycosylated CBMs in organisms capable of performing glycosylation, such as yeasts. The general aim of this project is to develop a novel approach to improve the properties of paper fibers using recombinant glycosylated CBMs. The yeast Pichia pastoris, which has proven to be an efficient and convenient system for large-scale production of heterologous glycosylated proteins, will be used as an efficient way to produce the glycosylated CBMs. The biological activity of the recombinant glycosylated CBMs will be characterised by interaction studies, using cellulose and lignin as substrates. The recombinant glycosylated CBMs, produced and purified in large-scale, will be subsequently used for the modification of pulp and paper properties, including recycled fibers. The improvement of drainability, associated to improved porosity during sheet formation (as demonstrated in our previous work through cake filtration assays) and higher hydration of the fibres are key properties which may be relevant namely in tissue papers production. The great advantage of our approach is that the recombinant glycosylated CBMs, which can be easily and rapidly obtained in high amounts, allow the substitution of cellulases/CBMs in papermaking, yielding similar results, but avoiding the undesired weakening of fibers, along with economic and environmental benefits. CBMs may be seen as a platform for modification of the surface properties of paper fibres. Although not in the scope of this project, but envisaged as a possible follow up, the association of CBMs with other proteins/molecules, by recombinant technology or chemical linkage, may provide valuable tools for the introduction of bioactive properties (e.g. using enzymes or antibodies) or labelling tags. The project success relies on the knowledge and commitment of a multidisciplinary team, composed by the IBB-UM and MTP-UBI patterns, whose complementary expertise (in the fields of molecular biotechnology and cellulose enzymology – IBB-UM - and paper technology – MTP-UBI) comprises all the subjects involving this proposal. The success of this project will not only contribute to a better making and finishing of industrial cellulose materials but also to a more economic and environmental-friendly processing.