Engineering enzyme-peptide fusion systems with self-assembly ability as advanced biocatalysts

Enzymes as biocatalysts are environmentally benign and their use is more sustainable compared to chemical catalysts, especially those containing toxic metals. The use of biocatalysts for industrial applications is increasing but are often used in their free and soluble form because they provide unimpaired activity while being simple and scalable in industrial settings, despite their limited reusability. Conventionally immobilised enzymes, those fixed to insoluble supports, can be reused in reactions but often with decreased activity while they attract additional costs of immobilisation. The ideal situation is therefore a system where biocatalysts have the best attributes of both the free and immobilised forms, high activity and recoverability, without significant increase in cost or difficulty in use.
 
   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.