Gruber, Pia; (2019) Challenges of Chemoenzymatic and Biocatalytic Reaction Cascades and Online Monitoring at a Microscale. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Microreactors have been established as useful platforms for chemical and biocatalytic reaction cascades. These are often difficult to implement due to substrate, product, solvent, cofactor, or pH-based inhibitions or unfavourable reaction equilibria, but the small dimensions and physical advantages of microreactors allow a cost-effective optimisation of such reactions. The implementation of sensors adds insight into the reaction progress and eases the reaction optimisation process. In this work, reactor designs, sensor integration protocols and reaction optimisation guidelines have been developed for two types of reaction cascades, namely chemoenzymatic and bi-enzymatic cascades. The chemoenzymatic reaction couples a Diels-Alder reaction with transketolase to produce 1-(3',4'-dimethylcyclohex-3'-enyl)-1,3-dihydroxypropan-2-one (DCDHP) and the two-enzyme cascade uses transketolase and transaminase to produce 2-amino-1,3,4-butanetriol. Optimisation of reaction conditions led to a 10 mM 2-amino-1,3,4-butanetriol production in two hours. The implementation of an aluminium chloride packed-bed reactor and use of acetonitrile as a solvent for the Diels-Alder reaction led to the production of 200 mM intermediate in only 50 min and a total process yield of 3.5 mM DCDHP in the coupled Diels-Alder – transketolase reaction after only 200 min. Sensors suitable for monitoring a range of pH 3.5-8.5 were developed to monitor reactions in which pH shifts occur due to product formation. Additionally, a carbon dioxide sensing system has been adapted for implementation into a microfluidic reactor to monitor the transketolase reactions used in both model systems, in which the side-product formation of carbon dioxide is used to drive the reaction. Finally, pH sensors were implemented into a microfluidic side-entry reactor to record a real-time pH profile at eight different locations in the reactor. This made manual adjustment of the pH in the reactor possible and resulted in a higher reaction yield. This shows that online monitoring can be used to improve reaction yields for enzymatic reactions at a microfluidic scale.

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