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Improving the use of asparaginyl endopeptidase for biocatalytic applications

Tang, Tsz Man 2021. Improving the use of asparaginyl endopeptidase for biocatalytic applications. PhD Thesis, Cardiff University.
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Asparaginyl endopeptidases (AEP) are cysteine proteases found in mammalian and plant cells. Several AEP isoforms from plant species were found to exhibit transpeptidase activity, which is integral for the key head-to-tail cyclisation reaction during the biosynthesis of cyclotides. AEPs isolated from plants exhibit excellent enzyme kinetics for peptide ligation via a relatively short substrate recognition sequence. Hence, AEPs could offer powerful applications as a biocatalyst in the production of modified proteins and peptides. However, the uptake of AEPs for biocatalytic application is limited by key challenges. In this thesis, an engineered AEP variant from Oldenlandia affinis, OaAEP1-C247A, is employed as a model enzyme to explore strategies which can address some vital limitations to the application of AEP catalysis. Fundamental enzymology of AEPs are discussed and a new protocol which simplifies the recombinant preparation of OaAEP1-C247A is described. To address the challenges posed by the reversible reactivity of the enzyme, a chemo-enzymatic approach to AEP-mediated peptide and protein bioconjugation is reported. Employing an affordable chemical reagent as a scavenger, this method takes advantage of the non-specific substrate recognition of OaAEP1-C247A. Accumulation of active proteases in the cytoplasm can be detrimental to the viability of the host cell. Therefore, strategies to encapsulate OaAEP1-C247A within a self-assembled protein capsid (AaLS-13) were explored. The caged protein complex serves as an artificial organelle for protein and peptide processing which mimics organelles and protein microcompartments found in nature. This synthetic biology approach offers the potential to enable AEP-catalysis in E. coli.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Date of First Compliant Deposit: 8 July 2021
Last Modified: 08 Jul 2022 01:30

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