Synthesis of single enzyme nanoparticles for enzyme stabilization

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Embargoed until 2023-10-12
Copyright: Wang, Yiping
Single enzyme nanoparticles (SENs), which encapsulate individual enzymes in a thin permeable polymer network offer great control over the chemical and physical environment directly around the enzyme. SENs have exhibited great enzyme stability by restricting enzyme extensive unfolding motion under extreme conditions, like extreme pH and high temperature. However, up to date, the control over the chemistry of the shell is still quite limited. In this thesis, a new SEN formation strategy has been explored. In order to minimize the risk of enzyme deactivation during synthesis of the SENs, the weak electrostatic interaction was utilized to assemble charged polymers around the enzyme. Different lengths of charged polymers were pre-prepared via reversible addition−fragmentation chain-transfer polymerization (RAFT) and then attached to the surface of enzyme via electrostatic interactions. This strategy has been investigated for the different enzyme, including lysozyme, trypsin, protease, horseradish peroxidase, and glucose oxidase. Isothermal titration calorimetry (ITC) and asymmetric flow field-flow fraction (AF4) in combination with multiangle light scattering (MALS) reveal the binding number and strength of polymer chains / enzyme. The strength of binding can be tuned based on the charge density of the bound polymer. In this method, the trithiocarbonate group of a RAFT agent was placed close to the surface of the enzyme and the initiation of a free radical acrylamide / bisacrylamide polymerisation in solution can result in chain extension of the RAFT polymer and direct the formation of the newly formed hydrogel around the outside of the enzyme. AF4-MALS and small-angle X-ray scattering (SAXS) confirm the formation of a thin cross-linked shell around the enzyme. The mild conditions of this method of SEN formation, which avoids any covalent modification of the enzyme, results in no loss in activity on our model enzyme (glucose oxidase), and four-fold increase in thermal stability. The method is then utilized to probe the protective effect of trehalose close to the enzyme. Trehalose is generally assumed to be the most effective sugar to use as a protein stabilizer. In this method, trehalose molecules were placed close to enzyme surface by either assembling enzyme with charged trehalose polymers or crosslinking with trehalose monomer. In order to evaluate the effect of the trehalose in SENs on stabilizing enzyme, another disaccharide sucrose was treated in the same way for comparison. It was found that the core-shell structure, instead of the chemistry of the shell, was more important for stabilizing enzyme structure under heat treatment. This study offers a new technique for synthesis of SENs with ease of design and control of the shell chemistry of SENs, opening up new pathways for enzyme stabilization and application.
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Wang, Yiping
Stenzel, Martina
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PhD Doctorate
UNSW Faculty