The Development of Continuous Microfluidic Immobilised Enzyme Reactor (μ-IMER) Systems and their Applications in Heterogeneous Biocatalysis

Enzymes and artificial metalloenzymes (ArMs) have been demonstrated in both research and industrial settings to be highly effective tools for achieving complex chemical transformations, such as enantioselectivity chemical synthesis, that would otherwise be highly inefficient or unobtainable via classical synthetic chemistry. Nevertheless, significant optimisation is required to develop these enzyme-catalysed reactions. Traditional approaches based on batch screening remain a highly labour-intensive, costly and time-consuming process. Using microfluidic immobilised enzyme reactors could address these limitations by enabling high throughput screening of the parameter space whilst reducing optimisation costs and time, with significantly lower volumes of precious chemical feedstocks and biocatalysts. However, little work within the literature compares different types of reactors for biocatalyst screening applications. This work focuses on developing packed-bed microcolumn and planar surface microfluidic chip reactor systems to compare and evaluate the performance and limitations of each. Two His-tag based surface immobilisation strategies were explored as part of developing the microfluidic chip reactor. First, an electrografting approach resulted in a dense molecular surface coverage of 3.29×1014 molecules cm-2, surpassing current literature values for similar processes. Second, a self-assembling monolayer based on thiol chemistries demonstrated an enzyme surface coverage of 6.86×1011 molecules cm-2. In order to minimise the amount, a novel continuous flow control system was created to automate control of the reactor setup. Successful inflow biocatalysis was demonstrated using a microcolumn reactor consisting of cystathionine beta-lyase (CBL) immobilised onto agarose beads to catalyse the α,β-elimination reaction of S-benzyl-L-cysteine at various concentrations. Although biocatalysis was not observed for CBL immobilised on a planar surface within a microfluidic chip reactor, the work explores the limitations of such techniques to provide grounds for furthering this area of research. The work additionally aims to contribute to the broader development of microfluidic chemical production lines for telescoped biocatalysis and enzyme cascade reactions.