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Title: Engineering robust enzymes via head-to-tail cyclization using spontaneous isopeptide bond formation
Author: Schoene, Christopher
ISNI:       0000 0004 6496 3623
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Enzymes are selective, operate efficiently in mild conditions and are biodegradable. Hence, enzymes have become an attractive option for a wide range of academic and industrial processes. However, enzymes are often labile at higher temperatures which can limit the operational range. Current enzyme stabilization approaches can be time-intensive and often have only a minor effect. Enzyme cyclization has been suggested as an alternative and more general approach. SpyTag/SpyCatcher, SnoopTag/SnoopCatcher and Isopeptag/Pilin-C are peptide/protein pairs capable of forming an irreversible isopeptide bond upon reconstitution. By genetically encoding SpyTag, SnoopTag and Pilin-C on the Nterminus and SpyCatcher, SnoopCatcher and Isopeptag on the C-terminus, I developed a method for the simple in vivo cyclization of enzymes. TEM-1 β-lactamase, which is prone to irreversible aggregation above 37 °C, remained soluble at temperatures up to 100 °C after isopeptide-mediated cyclization and upon cooling retained high catalytic activity. Differential scanning calorimetry showed that isopeptide-mediated cyclization had little effect on the Tm of the enzyme. Instead, isopeptide-mediated cyclization increased the reversibility of the denaturation. SpyTag/SpyCatcher was found to be the most effective pair for conferring thermal resilience and was also used to cyclize and increase the thermal resilience of dihydrofolate reductase and phytase. SpyTag/SpyCatcher-mediated cyclization had only minor effects on the thermal resilience of glucose oxidase and luciferase. Therefore, there is a limit to how general the effect of isopeptide-mediated cyclization is. To start addressing the limitations of the current approach, I developed a phage display-based strategy for the directed evolution of SpyTag/SpyCatcher reactivity. Isopeptide-mediated cyclization is a potentially powerful approach for the enhancement of protein resilience.
Supervisor: Howarth, Mark Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available