Browsing by Subject "degradation"
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Item Functional characterization of the chitin utilization system in the saprophytic bacterium Cellvibrio japonicus(2020-01-01) Monge, Estela C; Gardner, Jeffrey G; Biological Sciences; Biological SciencesChitin is a linear polymer of ? (1-4)-linked N-acetylglucosamine and is the second most abundant polysaccharide on earth. It is a major source of fixed carbon and nitrogen to microorganisms, and the strategies used by microbes to degrade chitin are of interest to the biotechnology industry as current chemical methods used are inefficient and wasteful. Due to the environmental and industrial relevance of chitin conversion the strategies used by microbes able to degrade this polysaccharide are of interest. Our current understanding of mechanistic and biochemical aspects of chitin degradation is fairly strong, however the physiological and genetic aspects of chitin degradation are not well characterized. The saprophytic Gram-negative bacterium Cellvibrio japonicus is a potent chitin degrader and has a suite of nine Carbohydrate Active enZymes (CAZymes) with predicted functions for chitin degradation: four chitinases from the Glycoside Hydrolase (GH) family 18, one GH19 chitinase, two GH20 hexosaminidases, one GH46 chitosanase, and one chitin-specific lytic polysaccharide mono-oxygenase (LPMO; Auxiliary Activity, AA10). My dissertations work had the goal to characterize the physiological roles of the chitinolytic machinery of Cellvibrio japonicus. During my dissertations work, I characterized the physiological roles of the C. japonicus GH18 chitinases during the degradation of chitin-rich substrates. I determined that the four GH18 chitinases (Chi18A, Chi18B, Chi18C, and Chi18D) have specific roles during the degradation of chitin and using a systems biology approach, and I determined that Chi18D is essential for chitin degradation. I also characterizated the mechanisms that C. japonicus employs to transport and metabolize chitin degradation products. Specifically, I functionally characterized the role of the two GH20 hexosaminidades in chito-oligosaccharide (CHOS) catabolism and I concluded that the gene product of hex20B has a major role in CHOS catabolism. Using transcriptomics and functional characterization I identified two TonB-dependent receptor genes cttA (CJA_0353) and cttB (CJA_1157). These two genes encode proteins essential for the transport of the products of chitin degradation. Furthermore, I characterized an operon for GlcNAc and GlcN transport and utilization by C. japonicus that has a glucosamine transporter and GlcNAc transporter. Finally, I developed a method to perform high-throughput screening of mutants using a custom designed 3D printed biocontainment devices, which allowed for the elucidation of subtle and complex phenotypes. I have not only characterized the physiological roles of the GH18 and the GH20 families of the saprophyte Cellvibrio japonicus but also proposed a model for chitin degradation in a soil bacterium. My dissertations expands our understanding of chitin degradation from an organismal perspective and validates the usage of multidisciplinary approaches to characterize the physiological roles of carbohydrate active enzymes.Item Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties(American Chemical Society, 2010-05-10) Zustiak, Silviya P.; Leach, Jennie B.The objective of this work was to create three-dimensional (3D) hydrogel matrices with defined mechanical properties, as well as tunable degradability for use in applications involving protein delivery and cell encapsulation. Thus, we report the synthesis and characterization of a novel hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel composed of PEG vinyl sulfone (PEG-VS) cross-linked with PEG-diester-dithiol. Unlike previously reported degradable PEGbased hydrogels, these materials are homogeneous in structure, fully hydrophilic and have highly specific cross-linking chemistry. We characterized hydrogel degradation and associated trends in mechanical properties, i.e., storage modulus (G′), swelling ratio (QM), and mesh size (ξ). Degradation time and the monitored mechanical properties of the hydrogel correlated with crosslinker molecular weight, cross-linker functionality, and total polymer density; these properties changed predictably as degradation proceeded (G′ decreased, whereas QM and ξ increased) until the gels reached complete degradation. Balb/3T3 fibroblast adhesion and proliferation within the 3D hydrogel matrices were also verified. In sum, these unique properties indicate that the reported degradable PEG hydrogels are well poised for specific applications in protein and cell delivery to repair soft tissue.