Enzymatic conversion of β-mannans: Analysing, evaluating and modifying transglycosylation properties of glycoside hydrolases

Retaining glycoside hydrolases are enzymes that catalyse the breakdown down of glycans through hydrolysis. Due to the double-replacement mechanism of the retaining glycoside hydrolases (GHs), which form an intermediate with part of the glycan covalently attached to the enzyme, some GHs are able to catalyse synthesis reactions called transglycosylation. In transglycosylation reactions a hydroxyl-containing molecule (acceptor), other than water, acts as a nucleophile which releases the glycan moiety from the covalent intermediate while forming a new glycoside (transglycosylation product). The transglycosylation reaction can be used to transform renewable starting materials such as plant hemicellulose to valuable products, which is discussed in the thesis. The work presented in the thesis have explored how GHs interact with glycans and how different aspects of transglycosylation reactions affect the final yield of transglycosylation products.

The presented work explores how the open active site structure of two GH26 β-mannanases have made them well adapted to act on heavily galactosylated hemicellulosic β-mannan polysaccharides (Paper I and II). In addition Paper I and II explore how substitutions of amino acids in glycan interacting subsites can lead to changes in catalytic properties and how the two GH26 β-mannanases productively interacts with oligosaccharides. The work also examines how variants of GHs can have improved transglycosylation capacity compared to their wildtype counterparts (Paper III and V). It investigates how the elimination of saccharide interactions in the +2 subsites can lead to improved transglycosylation capacity in a variant of the GH5 β-mannanase TrMan5A (Paper
III). The TrMan5A variant displayed greatly improved transglycosylation capacity at the early timepoints. Observed secondary (product) hydrolysis at later times highlighted the importance of analysing prolonged reaction times to determine suitable reaction termination. Paper III also demonstrated how enzyme synergy can lead to increased transglycosylation yields, when TrMan5A and a guar α-galactosidase was used in co-incubations where a galactomannan was used as the glycosyl donor. α-Galactosidases were further studied in Paper IV, where the
transglycosylation capacity of two different α-galactosidases were explored with different glycosyl donors and acceptor molecules. The study showed that the guar α-galactosidase was able to utilise a wide variety of acceptor molecules and glycosyl donors, further expanding potential transglycosylation products that may be produced with the enzyme. Paper IV further highlights the negative effects secondary hydrolysis may have on transglycosylation yields. The presented work also shows how targeting highly conserved residues within a glycoside hydrolase family can be used to quickly generate GH variants with improved transglycosylation capacity compared to the wild type GH (Paper V). The method relies on protein sequence data and does not require structural knowledge of the target enzyme. Furthermore, the method generates few variants (evolution (100s to 1000s) while it appears to be generally applicable as it was successfully applied to six different GH families covering varying specificities. Improvements was, in part, indicated to be associated with reduced secondary hydrolysis in several of the six GH families in the study.

The results presented in the thesis have expanded the knowledge of different factors that affects and can be manipulated in order to improve the transglycosylation capacity in retaining glycoside hydrolases. The work presented in the thesis will help further enzymatic synthesis approaches utilising renewable raw-materials.