Priprava in uporaba monolitnih nosilcev različnih mikrostruktur z imobilizirano glukozo oksidazo in hrenovo peroksidazo

Kupec, Rebeka (author), Podgornik, Aleš (mentor)

Monolitni nosilci se pogosto uporabljajo v afinitetni kromatografiji, kjer komponente ločujemo na podlagi bioloških interakcij. Kot ligande za substrat lahko pri tej tehniki uporabimo različne beljakovine. Pri tem se velikokrat odločamo za encime, saj ravno ti ob imobilizaciji dosegajo visoko ponovljivost eksperimentov, stopnja stabilnosti, ki jo dosežemo z imobilizacijo, pa jim omogoča dolgo življenjsko dobo. Učinkovitost vezave encima na monolitni nosilec je v prvi vrsti odvisna od narave encima (temperature, pH in ionske jakosti pufra, prisotnosti aktivatorjev, inhibitorjev in kofaktorjev), pa tudi od načina kovalentne imobilizacije in mikrostrukture nosilca. Monolitne nosilce smo funkcionalizirali z različnimi kemijskimi skupinami, da bi ugotovili, kako to vpliva na dostopnost encima glukoze oksidaze za substrat. Uporabili smo metakrilatne monolitne nosilce, funkcionalizirane z epoksidno, aldehidno in karbonildiimidazolno kemijsko skupino. Učinkovitost imobilizacije encima smo posredno ovrednotili z merjenjem absorbance, ki nastane zaradi vodikovega peroksida, produkta encimsko katalizirane reakcije glukoze. Eksperimentalni del smo izvedli na tekočinski kromatografiji visoke ločljivosti (HPLC). Iz dobljenih vrednosti smo izračunali hitrost encimskih reakcij, afinitetno konstanto ter pretvorbeno število in primerjali rezultate med monolitnimi nosilci.

• Language: Slovenian
• Keywords: monolitni nosilci, glukoza oksidaza, hrenova peroksidaza, encimska kinetika, afinitetna kromatografija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [R. Kupec]
• Year: 2019
• PID: 20.500.12556/RUL-107673
• UDC: 602.1:53.082.9:577.15(043.2)
• COBISS.SI-ID: 9215353
• Publication date in RUL: 12.05.2019

KUPEC, Rebeka, 2019, Priprava in uporaba monolitnih nosilcev različnih mikrostruktur z imobilizirano glukozo oksidazo in hrenovo peroksidazo [online]. Master’s thesis. R. Kupec. [Accessed 19 May 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=107673

Secondary language

• Language: English
• Title: Preparation and applications of monolith columns with different microstructural properties using immobilized glucose oxidase and horseradish peroxidase

Abstract

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.

• Keywords: monolith columns, glucose oxidase, horseradish peroxidase, enzyme kinetics, affinity chromatography