Bacterial biofilms, which are comprised of bacterial cells embedded in a thick extracellular polymeric substance (EPS), is a type of survival mechanism used by a variety of medically relevant bacteria to endure harsh conditions, the immune system of the host organism, and medical intervention such as antibiotics. Biofilms confer additional protection to these bacteria, protecting them from a number of stressors, and contribute to the growing problem of antibiotic-resistant infections. Biofilm EPS is comprised of extracellular polysaccharides, proteins, DNA, and other small molecules such as enzymes and nutrients, and the strength and structure of biofilms are often attributed to extracellular polysaccharides such as poly-β-D-(1→6)-N-acetyl-glucosamine (PNAG). Glycoside hydrolase enzymes that are produced as part of the biofilm’s life cycle are being explored as possible anti-biofilm compounds, due to their ability to destabilize biofilms through degradation of the polysaccharide components. The enzyme Dispersin B (DspB), a family 20 glycoside hydrolase produced by Aggregatibacter actinomycetemcomitans, hydrolyzes partially de-N-acetylated PNAG (dPNAG), and shows promise as a potential anti-biofilm agent. Here, we use a variety of techniques to investigate the interactions between DspB and PNAG, leading to a greater understanding of the binding interactions and mechanisms used by DspB to hydrolyze PNAG (Chapter 2). First, the activity of DspB on a monosaccharide probe, 4-methylumbelliferone-GlcNAc (4muGlcNAc) was observed over a pH range to determine the ideal conditions for DspB activity (2.2). Specifically acetylated PNAG trisaccharide analogs were then used to determine the substrate specificity of DspB, which supported the existing hypothesis that DspB uses a substrate-assisted mechanism to hydrolyze PNAG (2.4-2.6). These studies also indicated the possibility of electrostatic interactions between anionic amino acids on the binding surface of DspB and cationic deacetylated residues on PNAG that stabilize the substrate-binding interactions and allow for additional cleavage activities of DspB, namely improved cleavage of partially deacetylated PNAG and the ability to perform endo- or exoglycosidic cleavage activity, dependent on the substrate acetylation patterns present (2.5). Mutagenesis of amino acid residues on the binding surface of DspB was performed to investigate these interactions (Chapters 3-4), resulting in the discovery of an improved DspB mutant. This E248Q mutant of DspB also has an improved ability to clear Staphylococcus epidermidis biofilms, indicating that it may have improved anti-biofilm activity (3.3). Finally, a high-throughput assay for anti-PNAG activity has been developed for use with a degenerate DspB mutant library in order to identify additional DspB mutants with improved anti-biofilm activity.