Saunders, Jake2023-11-082023-11-08http://hdl.handle.net/1885/305642Enzymes have become vital in many industries, from chemical synthesis to food production and textile manufacturing. However, many enzymes are commonly limited by low substrate specificity or a lack of stability, making them rarely suited to industry applications. Protein engineering, often aided by computational algorithms, aims to modify the genetic code to alter the structure and function of an enzyme. This field has seen dramatic growth in the past few decades, showing success in many different applications, including the creation of new biosensors, enzymes used in chemical and pharmaceutical production, and improved enzymes for plastic degradation. Although still in its infancy, this field has yet to reach its true potential. This thesis uses various engineering techniques to improve several enzymes. Following the introduction, the first part of the thesis follows the protein engineering story of enzymes involved in the degradation of PET plastic, specifically MHETase and PET hydrolysing cutinases. The latter part of this thesis focuses on using the bioinformatics and protein engineering technique ancestral sequence reconstruction to design variants of the asparaginyl endopeptidase (AEP) family and N2-(2-carboxyethyl)-arginine synthase (CEAS) that hopefully possess enhanced properties. Chapter 1 introduces the critical information and concepts that this thesis revolves around, providing insight into the protein engineering tools used herein. Chapter 2 explores the protein engineering of MHETase, first characterising a new assay method to detect this enzyme's activity and then using this assay to develop a highly expressible variant to aid in future engineering projects. Chapter 3 investigates the impact that point mutations identified from ancestral sequence reconstruction (ASR) have on the structure of a PET hydrolyse cutinase. Chapter 4 provides a comprehensive overview of the current state of ASR in its applications in protein engineering. Chapter 5 explores the practical application of ASR to identify new variants of the plant AEP family with improved or modified functionality to generate an improved peptide ligation tool for protein and peptide synthesis and engineering. In Chapter 6, we examine the use of ASR on CEAS, an enzyme that exhibits a distinctive catalytic intermediate, to identify ancestors and their mutations that may improve enzyme stability or promiscuity to streamline the chemical synthesis of B-lactone derived antibiotics. Finally, Chapter 7 concludes the thesis by bringing together all the ideas discussed throughout this thesis and discussing recent developments around this project and future aspirations.en-AU202310.25911/BCK7-FM86