UBC Theses and Dissertations
Heterologous production and functional characterization of Auxiliary Activity family 10 lytic polysaccharide monooxygenases for fiber functionalization Li, James
The efficient depolymerization and/or utilization (valorization) of residual waste biomass is crucial for establishing a sustainable bio-based economy. Cellulose and chitin constitute the most abundant polymer components of biomass and represent renewable feedstocks for the production of biofuels, biomaterials, and other bio-based chemicals. The recalcitrance and low chemical reactivity of these polysaccharides limit their viability in numerous industrial applications. Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes secreted by saprotrophic organisms to assist in the depolymerization of cellulose and chitin fibers. The recent discovery of LPMOs has shifted the fundamental understanding of microbial biomass utilization while also potentiating many avenues for the commercial valorization of cellulose and chitin. This dissertation presents a thorough investigation of the production, functional analysis, and fiber-modifying application of bacterial LPMOs from Auxiliary Activity family 10 (AA10). Signal peptide-mediated periplasmic secretion through the general secretion pathway (Sec) in Escherichia coli was the most suitable approach for producing active AA10 LPMOs. Five AA10s encoded by the historically significant Cellulomonas flavigena and Cellulomonas fimi bacteria were purified and characterized. Four of these Cellulomonas AA10 homologs were cellulose-active and one was chitin-active. Thermostability studies confirmed only cellulose-active Cellulomonas AA10s could reversibly unfold. Three non-Cellulomonas AA10 catalytic domains were produced in E. coli using the Sec signal peptide derived from the lone AA10 encoded by C. fimi. Notably, Xylanimonas cellulosilytica LPMO10A demonstrated dual activity on cellulose and chitin, while Streptomyces pratensis LPMO10D and Legionella longbeachae LPMO10 were found to be strictly active on chitin or cellulose substrates, respectively. Time course kinetic assays show high initial LPMO-oxidizing activity commonly coincides with rapid inactivation from oxidation. These results highlight the importance of optimizing abiotic parameters such as pH and reductant type to maximize the catalytic potential of LPMOs. Lastly, as a proof-of-concept, a C1-oxidizing cellulose-active AA10 encoded by C. flavigena was used to chemo-enzymatically conjugate several fluorescent probes to cellulose fibers. The work presented here informs future studies on heterologous LPMO production, LPMO activity screening and characterization, as well as LPMO-mediated fiber defibrillation and chemical modification.
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