All cells and extracellular matrices of prokaryotes and eukaryotes are made up of glycans, the carbohydrate macromolecules that play a predominant role in cell-to-cell interaction, protection, stabilization, and barrier functions. Glycans are also central to human microbiome-host interactions where bacterial glycans are recognized by innate immune signaling pathways, and host mucins are a major nutrient source for various gut bacteria. Many microorganisms encode glycoside hydrolases (GHs) to utilize the available host cell surface glycans as a nutrient source and to modulate host protein function. The GHs are divided into families having conserved linkage specificity within each family and individual family members can be specific for dramatically divergent macromolecular substrates. In general, within a given GH family very few members have been biochemically characterized and the substrate specificity is poorly understood. GH genes are abundant in the human gut microbiome and culture-enriched metagenomics identified more than 10,000 distinct bacterial GH genes in an individual. The focus of this thesis is endo-β-N-acetylglucosaminidases (ENGases) encoded by GH18 and GH85 families. Bioinformatic analysis shows that the predicted proteins within each of these GH families fell into separate clusters in the Sequence Similarity Networks of each family. The hypothesis of this project is that human microbiome-encoded ENGases from the same GH family differ in their substrate specificities and within the SSN network of the same GH family, enzymes with similar substrate specificity may fall in the same cluster. In this work, I established conditions for overexpression of GH18 and GH85 proteins and investigated the activity of these enzymes on various substrates. / Thesis / Master of Science (MSc) / All the cell surfaces of animals, plants, and microbes are coated with sugars, also known as glycans. These sugars on the cell surface act as a barrier and protect them from the external environment. Glycans on the cells of both microbes and humans are essential for basic interactions between them. Many bacteria produce enzymes such as glycoside hydrolases to obtain nutrients from dietary sugars and alter the sugars on host proteins. There are various families of these enzymes, and they act on specific sugars and cleavage sites. The substrate specificities and characterization of these enzymes from most bacteria found in the human microbiome have not been studied in detail. My work focuses on developing standard enzyme assays for determining specific substrate specificities. This tool can be used to reshape glycans and understand their role in cell processes.