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Engineering enzyme-peptide fusion systems with self-assembly ability as advanced biocatalysts
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
This thesis focusses on engineering enzymes with a self-assembly feature to form functional enzyme particles. The engineered self-assembly ability allows controlled immobilisation of enzymes, minimising loss of enzyme activity, while eliminating the need for solid-supports. This carrier-free approach minimises both the mass-transfer limitations and cost of enzymes that are immobilised to solid carriers. However, self-assembly is not an intrinsic property of enzymes and requires a partner molecule to confer this feature. For this purpose, we have chosen a self-assembling peptide as a partner molecule for enzymes, serving as a novel immobilisation approach.
The research comprises four major experimental sections that range from proof-of-concept and understanding the self-assembly mechanism to examining enzyme reusability and exploring the assembly approach as a platform technology for engineering reusable enzyme particles. Using bovine carbonic anhydrase (BCA) as a model enzyme and P11-4 peptide as the assembly partner, the first experimental section demonstrates the concept of enzyme-peptide self-assembly into nanoparticles, followed by evaluation of enzyme activity and its application for CO2 capture. The second section investigates the mechanisms that control the self-assembly process of the BCA-P114 model system. Key factors such as effect of pH, temperature, salts etc. have been systematically examined to reveal their influence on self-assembly of enzyme, demonstrating that metal-ions and pH change can work independently, or in combination, to trigger self-assembly. An empirical model was developed that predicts the particle size under different solution conditions allowing for a tunable enzyme-peptide particle of desired size.
The third section studied the effect of additional peptide units on the self-assembly and activity of the enzyme-peptide and reusability of formed particles. A long peptide containing 3 repeats of P11-4 peptide was compared with a single P11-4 peptide, showing that the addition of extra repeats alters the self-assembly structure of the enzyme from nanoparticles to resoluble aggregates. Importantly, both BCA-P114 and BCA-(P114)3 systems were demonstrated to be reusable, having been retained using ultrafiltration and precipitation, respectively. The fourth section explores self-assembly of a range of industrially-important enzymes. Four enzymes, each from a different class, were selected and fused with a single P11-4 peptide, followed by systematic evaluation of their recombinant production in Escherichia coli, purification, self-assembly and activity compared with the wild-type enzyme without peptide. The outcome demonstrated the assembly method as a platform technology to engineer enzyme particles using a variety of industrially important enzymes.
In conclusion, a novel immobilisation approach for enzymes has been established, based on the self-assembly feature of a designed peptide. The discovery and investigation of the engineered biocatalysts has provided the fundamental knowledge to guide development of carrier-free reusable enzyme particles without additional cost.