Monolith columns are frequently used in separation methods of biological components in affinity chromatography. Such columns often serve as carriers for various proteins, which interact with substrates. In this matter, enzymes are among most commonly used proteins, since they ensure repeatability of measurements, and are highly stable. The efficiency of binding between enzyme and monolith column depends on chemical properties of enzyme (temperature, pH, ionic strength of buffer solutions, presence of activators, inhibitors and cofactors), type of covalent binding, and monolith microstructure. The emphasis of this Master thesis was to evaluate glucose oxidase activity at different glucose concentrations while the enzyme was immobilized on metacrylic monoliths through different functional groups: epoxide, aldehyde and carbonyldiimidazole group. Enzyme activity was evaluated indirectly by measuring the absorbance, caused during formation of hydrogen peroxide which is the end product of enzyme reaction with glucose. The experimental part was concluded on High performance liquid chromatography equipment (HPLC). Results were used to calculate and compare the affinity constant, turnover number and maximal velocity rate of the enzyme for each sample with its own functional group.
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