Exploration and discovery of enzymes and non-catalytic proteins targeting microbial α-glucans

Doctoral thesis, 2026

Polysaccharides are everywhere. Many have structural or energy-storage roles, such as the well-known plant polysaccharides cellulose and starch. This thesis focuses on the less studied α-1,3-glucans, which can be found in fungal cell walls and bacterial biofilms fulfilling various functional roles. In fungal cell walls, α-1,3-glucans are known to function as a storage polysaccharide in some species, and as virulence factor in others. In dental plaque, α-1,3-glucans build up the protective structure and provide adhesion to the tooth, contributing to the characteristic stickiness and resilience of biofilms. Because of their structural importance and involvement in pathogenicity, there is interest in the degradation of α‑1,3-glucan. The aim of my thesis studies was to increase the knowledge concerning which enzymes can degrade α‑1,3-glucan (mutan) and mixed-linkage α-1,3/α-1,6-glucan (alternan) and how these enzymes function.

I have explored the two carbohydrate-active enzyme families currently known to contain α‑1,3-glucanase activity: glycoside hydrolase families (GH) 71 and 87. Two GH71 enzymes from the fungus Aspergillus nidulans were studied. Through structural determination coupled with biochemical characterization we elucidated the mechanisms and catalytic amino acids for these enzymes. I also studied a GH87 enzyme from Flavobacterium johnsoniae and discovered that one of its appended domains is a novel carbohydrate-binding module. By binding both mutan and alternan, its presence significantly improves the activity of the enzyme compared to the lone catalytic domain.

I also discovered new sources of mutan- and alternan-degrading enzymes via screening of environmental soil samples to identify microorganisms able to grow on these polymers. Six Streptomyces isolates were chosen for further study, which led to the description of two new species, Streptomyces castrisilvae and Streptomyces glycanivorans. In addition, analysis of the secretome of two of the isolates during growth on mutan and alternan showed that both strains employed several GH87 enzymes, all with different domain architecture, for the degradation of both polysaccharides. Growth on alternan also induced the expression of a gene cluster whose proteins’ concerted actions were shown to facilitate alternan and dextran (α-1,6-glucan) utilization.

Overall, my work contributes to the understanding of carbohydrate-active enzymes and carbohydrate-binding modules targeting microbial α-1,3-glucans.