Hybrid materials - Synthetic biology approaches to design functional structures

Abstract

Producing functional quantities of soluble and active eukaryotic lysyl oxidase has been a longstanding goal for research and industry. Herein, studies are reported of an archaeal LOX (MBP_HT_LOX) that overcomes longstanding issues of recombinant overexpression in E. coli cells. The purification and biophysical characterisation of enzymatically active MBP_HT_LOX is reported through an HRP-coupled absorbance reporter system with cognate and industrially relevant substrates, which influenced the bioengineered design of a reductionist ϵ-Polylysine based hydrogel suited for our enzyme. The action of MBP_HT_LOX to initiate covalent imine bonds was analysed through SAXS, and in our hydrogel system, as changes to morphology and viscoelastic properties monitored through SEM, FTIR and rheology. These investigations provide the first evidence of non-mammalian lysyl oxidase activity, both in vitro and ex vivo, as a biological crosslinker and can be incorporated into composite materials to act as a healing and rigidification agent that is non-toxic to human cells. In complimentary studies, bioinformatic analysis highlighted potential thermophilic actinomycete LOX homologues (AmLOX) with increased solubility, stability and thermal catalytic performance. Mutagenesis experiments guided by structural models and molecular dynamic simulations improved the solubility and purification of enzymatically active AmLOX. The effect of temperature on the kinetic rates of these enzymes was evaluated using macromolecular rate theory (MMRT), which revealed a change in heat capacity between mutants accounts for the curvature of kinetic rates with increasing temperature below the melting temperature.

Date of Award	22 May 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	J L R Anderson (Supervisor) & Paul Race (Supervisor)