Browsing by Subject "biocatalysis"
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Item Engineering Biocatalytic Materials: Encapsulation Systems for Biotechnology(2018-09) Sakkos, JonathanMy dissertation presents scientific and engineering contributions. Both sol-gel and layer-by-layer technologies were used to study prokaryotic cells under confinement and the role that the encapsulation material plays in affecting these cells. My work focused on the development, synthesis, and use of biologically active materials for water treatment based on silica gel bioencapsulation. Silica gels were developed with improved mechanical properties while ensuring that these materials were cytocompatible. The ratio of the silicon alkoxide crosslinker to silica nanoparticles was crucial in adjusting the maximum stress at failure. I demonstrated a 6-fold improvement in the compressive stress at failure in a silica bioencapsulation matrix containing metabolically active cells. These gels were organically modified to study the effect of encapsulation matrix hydrophobicity on the relative adsorption and biodegradation of organic pollutants. The ratio of biodegradation to adsorption was a strong function of the hydrophobicity of the pollutant. A layer-by-layer approach was used to minimize the diffusion length and protect the encapsulated biocatalyst from environmental stressors. I showed that targeting the cytoplasmic membrane with detergents increased its permeability and enabled a 15-fold enhancement in the rate of biocatalysis. To demonstrate the protective effects of the microbial exoskeleton, the coated cells were exposed to environmental stressors ranging from heat shock and desiccation to enzymatic attack and predation by protozoa. With a minimum of 4 layers, the biocatalytic activity was preserved for all cases examined. The translational application of cyanuric acid hydrolase (CAH) was also studied. By treating the cells with glutaraldehyde to crosslink the membrane-bound proteins, the cell was stabilized and CAH was retained within the whole cell biocatalyst.Item Isolating and Assaying Unspecific Peroxygenase and Flavin Binding Enzymes for in vitro Terpenoid Biosynthesis(2018-05) Hanson, BenjaminTerpenoids are an exceptionally large family of natural products, and contain numerous bioactive members that are pharmaceutically important. While most research into terpenoids and their metabolism has thus far occurred in non-fungal organisms, chiefly plants, Basidiomycota (mushroom forming fungi) are well known as prolific producers of bioactive sesquiterpenoids, such as the potent anticancer compounds Illudin M and S. While natural products have traditional been a huge driver of pharmaceutical discovery, this natural abundance is often hampered by very low expression in the native host and slow growth or rarity of the host itself. This drives up financial and environmental costs, and in many cases makes the production of otherwise useful natural products impracticable. One potential solution to this quandary is heterologous production of secondary metabolites in non-native hosts such as Escherichia coli and Saccharomyces cerevisiae. Another avenue is in vitro biocatalysis, wherein the biosynthetic enzymes of the native host are heterologously expressed, isolated, and used to perform synthesis outside of the cell. This approach avoids the fragility of the in vivo system and would allow the creation of combinatorial enzymatic pathways to create novel bioactive structures. With regards to constructing a sesquiterpenoid biosynthetic pathway, many stable terpene synthases have been isolated and shown to be active in vitro. However, the most well studied terpene scaffold modifying enzymes, cytochrome P450s, are notoriously difficult to heterologously express in active form. In order to obtain scaffold modifying oxygenases capable of being part of an in vitro terpenoid biosynthetic pathway, fungal oxygenases aside from cytochrome P450s were investigated. Specifically, unspecific peroxygenase from Agrocybe aegerita and flavin binding oxidoreductases from Δ6-protoilludene biosynthetic gene clusters were expressed and assayed against the sesquiterpene scaffold.