Cellodextrin and β-D-1,3-glucan phosphorylases as biocatalysts for novel glucan structure synthesis

Enzymatic synthesis of polysaccharides is a relatively new field of science which combines innovative materials with the biological precision of enzymes. The publications presented in this thesis demonstrate that enzyme-catalyzed reactions can be utilized to produce unique carbohydrate-based materials. The work focuses on enzymatic synthesis of cellulose and β-1,3-glucan with phosphorylase enzymes, aiming at the same to influence the structural properties of the produced polysaccharides for example by adjusting reaction conditions and performing the reactions with the enzyme's native and non-native glycosyl acceptors. Publication 1 focuses on a recombinantly produced cellodextrin phosphorylase from Clostridium thermocellum bacteria and its application in in vitro cellulose synthesis. The most relevant findings of this work were that the length of the synthetic cellulose polymers as well as their structural properties of the cellulose fibrils that formed, could be influenced based on the initial concentration of the glycosyl acceptors. These results lead towards tailored cellulose materials that can be used in different applications. Utilizing similar methodology, in publication 2 we investigated β-1,3-glucan synthesis with a recombinantly produced β-1,3-glucan phosphorylase. When the synthesis reactions were carried out at certain temperatures, unique layered hexagonal particles were produced. These results improve our understanding on the structural behaviour of triple-helical β-1,3-glucans and broaden the range of enzymatically synthesizable carbohydrate-based structures. Publication 3 broadens the scope of polysaccharide synthesis by utilizing chromophoric glycosyl acceptors as substrates for enzymatic synthesis reactions, which makes it possible to attach color molecules covalently to the structures that are formed as a product. This approach adds color molecules to the list of application areas for enzymatically synthesized materials and improves their attractability. Together, this research improves our understanding on the mechanisms of phosphorylase-catalyzed polysaccharide synthesis and leads towards tailored biomaterials. The implications of this research are far-reaching, and they have potential applications in smart materials, biocompatible and functional materials among other. This thesis highlights the broad potential of glycoside phosphorylases in biomaterial science and lays the groundwork for developing tailored carbohydrate-based materials.