Biocatalysts represent a green alternative to traditional catalysts, as they offer high selectivity and stereospecificity, while enabling operation under milder conditions and in environmentally friendly solvents. Enzyme immobilization allows easier separation from the reaction mixture, reuse, and improved stability – properties desirable in industry, where the broader application of biocatalysts is often limited precisely by their low stability and high production costs. By carrying out biocatalytic processes in microflow systems, the consumption of biocatalysts, solvents, and reactants can be reduced, while rapid heat and mass transfer enables process intensification.
In this master’s thesis, we investigated the tyrosine decarboxylase MfnA from the thermophilic archaeon Methanocaldococcus jannaschii, which catalyzes the conversion of L-tyrosine to tyramine. Since the enzyme functions at elevated temperatures, we encountered and successfully addressed practical challenges related to performing reactions under such conditions.
First, we determined the influence of temperature, buffer, and the concentrations of substrate, product, and the cofactor pyridoxal-5′-phosphate (PLP) on enzyme activity. We then evaluated different approaches to enzyme immobilization: on porous polymeric carriers (ReliZyme™ EP112/S and EA403/S), on functionalized cellulose membranes, and in the form of cross-linked enzyme aggregates (CLEA). In this context, we investigated the effects of enzyme loading, pH, and the concentrations of precipitant and cross-linker on immobilization efficiency. The immobilized preparations were also used in flow microreactors for continuous biotransformation.
The results showed that MfnA is strongly prone to aggregation upon dilution, and therefore we were unable to prepare CLEAs with favourable properties using standard procedures in the microfluidic system. Immobilization on porous polymer carriers ReliZyme™ EP112/S proved to be the most successful, achieving 42.7 % recovered activity, with the enzyme remaining stable for at least one week. Using the immobilized enzyme in a flow microreactor, we achieved 100 % product yield, demonstrating the potential of synthesis in a microflow system with an immobilized enzyme for continuous biotransformations.
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