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theses

Designer oligosaccharides can be used as prebiotics for selective bacterial growth and are therefore relevant to understanding how complex biological systems function in a nutrient-limited environment. Enzymatic synthesis offers an alternative route to producing designer oligosaccharides with higher reaction specificity, product purity, and lower production costs compared to standard chemical synthesis routes. Here, we focus on glycosyl hydrolase (GH) enzymes from GH family 29 that were reverse engineered to function as glycosynthase enzymes for synthesis of fucosylated oligosaccharides. First, we used two distinct model fucosylated oligosaccharides as a nutrient carbon source to highlight how targeted growth of bacteria is feasible for bacterial species expressing the wild-type GH 29 enzymes over bacterial species not expressing the relevant gene of interest. This proof-of-concept experiment helped clearly establish how designer oligosaccharides could be used to modulate the growth of specific bacteria from a complex microbial milieu. Next, detailed bioinformatics analyses and mechanistic assays of two GH family 29 enzymes were carried out to study the impact of certain active-site nucleophilic site mutations on glycosynthase activity using model substrates. Here, we specifically focused on GH29 enzymes with known distinct substrate preferences belonging to Bifidobacterium longum subspecies infantis or Blon_2336 (ACJ53394.1) and Thermotoga maritima or Tm_0306 (AAD35394.1). Additional molecular docking simulations were carried out to provide a clear rationale for why certain GH29 family of enzymes are able to selectively hydrolyze certain isomers of fucosyllactose to facilitate selective bacterial growth. Lastly, molecular docking simulations were conducted to identify other novel mutation sites to the native GH29 enzyme that would be necessary to improve fucosylated oligosaccharide synthesis efficiency using engineered glycosynthases. In summary, this study provides a clear rationale for how distinct carbohydrate-based oligosaccharides, can be synthesized chemoenzymatically using a rationale structure-guided GH enzyme engineering approach, and how these distinct carbon sources can be used to selectively target growth of certain bacterial species carrying the relevant GH genes of interest.

ETD_9339

M.S.

Includes bibliographical references

by Namratha Subhash

doi:10.7282/t3-f50x-dw82

ETD graduate

The author owns the copyright to this work.