Biocatalytic valorisation of natural polymers

Abstract

Biocatalysis is the rapidly-expanding field of enzymes applied as catalysts in chemical reactions. Biocatalysts are used by the pharmaceutical industry to produce a range of clinically available drugs, they also find use in consumer products e.g. detergents. Biocatalysis continues to increase in popularity since it meets various green/sustainable criteria that includes reduction of fossil fuel feedstocks and the creation of a more environmentally conscious market. Biocatalysts work at low temperature, in water, as opposed to organic solvents. They also display high enantio-, stereo- and chemo- selectivity limiting side products. The plethora of biocatalyst-driven chemical reactions continues to grow and can be engineered/optimised by directed evolution and selection.

This thesis describes work in the application of biocatalysts in the industrial biotechnology sector. An overall goal is to increase the value of natural polymers sourced from the wool and agricultural industries some of which are currently deemed as “waste” materials with low commercial value. The aim is to apply biocatalysts to the treatment of wool fibres and vegetable-derived cellulosic material (Curran®). Wool consists of -keratin where loose helical structures are covered in scales that contribute to the larger, coarse fibre. This study describes efforts to make these fibres more appealing to consumers by the development of a method for the controlled degradation that leads to the softening of keratin by keratinase biocatalysts. It begins with a review of the literature to select proteinase candidates with keratinase activity. Two commercial proteinases (Proteinase K; PK and Subtilisin Carlsberg; SC) were used to develop a colourimetric (λ = 595 nm) keratinase assay using keratin azure, a commercial anthraquinone blue dyed wool. This sensitive, robust, reproducible method was calibrated against the dye standard. This assay was also used to measure the activity of recombinant keratinase candidates which resulted in a preferred proteinase, Brevibacillus sp. WF146, being taken forward for further development. Recombinant WF146 was expressed and purified from E. coli both at small and larger scales. The resulting biocatalyst was applied to ‘raw’ sheep wool where the degradation was observed by light microscopy.

The most abundant polymer which is a commonly “wasted” resource is the cellulosic material. Cellulose can be modified using chemical reagents creating different properties i.e. hydrophobicity. Current methods use strong toxic/corrosive chemicals so this project investigated alternative, biocatalyticdriven methods acting on the sugar beet-derived cellulose, Curran®. The strategy used model substrates (glucose, cellobiose and cellulose) before application to the industrial material, Curran®. The most promising route was identified as esterification which was catalysed by commercial and recombinant lipases. A recombinant lipase, Thermobifida fusca (TfCut2) purified from E. coli, was chosen since it has a large, open catalytic active site which could accommodate polymers. The TfCut2 biocatalyst could esterify glucose and cellobiose in water and this was optimised from a cellulose source through green solvents. By combining the TfCut2 with a commercial mixture cellulases, CTec2, it was possible to break down cellulose to glucose and convert it to high value glucose monoesters. These esters have applications in the food, cosmetic and drug delivery industries.

Overall, this thesis describes successful efforts in identifying and screening different biocatalysts with potential applications in the valorisation of two highly abundant natural polymers.