Biocatalysis holds significant potential in the field of green and sustainable chemistry, as it enables reactions under mild conditions, such as physiological pH, moderate temperature, and pressure. Additionally, it allows for high reaction yields and stereoselectivity of products. Biocatalytic syntheses are shorter, more energy-efficient, and less environmentally burdensome compared to traditional organic syntheses. Despite these advantages, commercialization is often hindered by the lack of enzyme stability during operation and their relatively high cost. One solution to these limitations is enzyme immobilization, which enhances their stability, simplifies separation after the process, and allows for reuse. Immobilizing enzymes in cross-linked enzyme aggregates (CLEAs) using microfluidic systems has proven to be a promising technique, enabling repeated and prolonged enzyme use in continuous systems.
Glucose oxidase is an enzyme that catalyzes the oxidation of β-D-glucose with molecular oxygen into D-glucono-δ-lactone, which subsequently hydrolyzes spontaneously into gluconic acid. It is naturally produced by certain fungi and insects, where its catalytic product, hydrogen peroxide, acts as an antimicrobial agent. Due to its high electrochemical activity, it is an important component of glucose sensors and has potential applications in fuel cells.
In this master’s thesis, we optimized a microfluidic system for synthesizing CLEA particles of selected glucose oxidase with uniform size and high retained activity. We investigated how synthesis concentrations affect particle activity and size. Optimization was performed using the Box-Behnken experimental design method, examining three variables (enzyme concentration, precipitant concentration, and cross-linker concentration) at three levels.
The results showed that we successfully produced uniform particles with an average hydrodynamic radius of 113 ± 1.37 nm and a 100 % immobilization yield. Additionally, as part of this research, we achieved a recovered enzyme activity of 91.39 ± 1.68 %, indicating the potential applicability of this technique in industrial processes.
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