Interest in biocatalysts is increasing as they catalyse reactions under mild conditions and often provide enhanced control surrounding regio-, stereo- and enantio-selectivity. Yet, their use within the pharmaceutical industry is limited compared to regular chemocatalysts. Advances in protein engineering strategies are increasing the suitability of biocatalysts within industrial processes. However, these strategies produce huge variant libraries (10^6 -10^8 is typical) and the availability of the high-throughput screening methods to rapidly assess the mutants fitness is currently lacking, ultimately slowing their development. Raman spectroscopy is a technique offering molecular specific information and is highly suited towards aqueous conditions, thus presenting itself as attractive alternative screening method. Raman scattering is inherently weak, thus enhancement techniques, such as surface-enhanced Raman scattering (SERS) and ultraviolet resonance Raman (UVRR) spectroscopy, can enhance the weak response. Discussed within, we successfully implement SERS and UVRR to monitor a wide range of biotransformations. Firstly, the regioselective activity of flavin-dependent halogenases (FDHs) were investigated using SERS. These studies identified that SERS could distinguish between regioisomeric products. However, the complexity of the biotransformation, involving essential cofactors, caused significant problems in the detection of substrate and product, rendering this approach unsuccessful. Next, we developed thiol-functionalised substrates to combat competition experienced by the cofactors. Exploiting sulfurs high affinity towards silver proved to be extremely successful, resulting in a negligible cofactor response. However, FDHs did not display activity towards the sulfur-containing substrates, thus, we were unable to monitor activity. Furthermore, we implemented this thiol-functionalisation approach to effectively distinguish between the enantioselectivity of a protease: chymotrypsin. Synthesis of novel pseudo-enantiomer substrates, incorporating different thiol-linkers, led to easy, real-time discrimination of enantioselectivity using SERS. Lastly, an on-line UVRR approach was developed which allowed accurate and reproducible measurements of nitrile-hydrolysis, mainly nitrile hydratase. Additional development of a flow-cell set-up and introduction of whole-cells further established its aptness towards industrial processes, such as process analytical technology (PAT). The research presented within this thesis highlights the suitability of Raman enhancement techniques to rapidly analyse biotransformations, in some cases real-time measurements have afforded accurate and reproducible quantitative analysis.
|Date of Award||1 Aug 2018|
- The University of Manchester
|Supervisor||Jason Micklefield (Supervisor) & Royston Goodacre (Supervisor)|