| Popis: |
Namen magistrskega dela je bil s koprecipitacijo Fe2+ in Fe3+ ionov sintetizirati magnetne nanodelce, jih modificirati z dekstranom in proučiti njihovo uporabo kot nosilcev za imobilizacijo encima transglutaminaza. Sintetizirane nanodelce smo najprej aktivirali z mrežnim povezovalcem GA in/ali PEHA in jih nato uporabili za kovalentno imobilizacijo encima. Določali smo učinkovitost imobilizacije in aktivnost proste ter imobilizirane transglutaminaze. Proces imobilizacije smo optimirali s spreminjanjem koncentracije mrežnih povezovalcev (GA, PEHA), časa imobilizacije, koncentracije encima, koncentracije stabilizacijskih proteinov (EA, BSA), temperature imobilizacije, hitrosti stresanja in s spreminjanjem zaporedja dodajanja stabilizacijskih proteinov ter vrste nosilca. Določali smo tudi termično stabilnost encima in možnost ponovne uporabe. Rezultati kažejo, da so dekstranski nanodelci primeren nanonosilec za imobilizacijo transglutaminaze, saj smo pri optimalnih pogojih imobilizacije (aktivacija nosilca z 10 vol. % PEHA, stabilizacija encima s 50 mg/mL BSA, 100 mg/mL TGM, 4-urna imobilizacija pri 10 °C in 400 obr/min) dosegli hiperaktivacijo encima in s tem 99 % učinkovitost imobilizacije ter 110 % preostalo aktivnost encima. Hiperaktivacijo encima smo zaznali tudi z imobilizacijo encima na dekstransko-oleinske nanodelce. Imobiliziran encim je bil stabilen pri višjih temperaturah, prav tako je bila možna tudi njegova ponovna uporaba. The purpose of the master’s thesis was a synthesis of magnetic nanoparticles by the coprecipitation of Fe2+ in Fe3+ ions, their modification with dextran, and an examination of their use as carriers for immobilization of the enzyme transglutaminase. Synthesized nanoparticles were first activated with a cross-linking agent (GA and/or PEHA) and then used for covalent immobilization of the enzyme. The effectiveness of immobilization and the activity of free and immobilized transglutaminase was determined. The process of immobilization was optimized by changing the concentration of cross-linking agents (GA, PEHA), the time of immobilization, enzyme concentration, the concentration of stabilizing proteins (EA, BSA), immobilization temperature, agitation rate, type of carrier, and by changing the sequence of adding stabilizing proteins. The thermal stability and reusability of the enzyme were also investigated. The results indicate that the dextran nanoparticles are a suitable nanocarrier for the immobilization of transglutaminase, since under optimal immobilization conditions (activation of the carrier with 10 vol. % PEHA, stabilization of the enzyme with 50 mg/mL BSA, 100 mg/mL TGM, 4-hour immobilization at 10 °C and 400 rpm) the enzyme was hyperactivated and shows 99% immobilization efficiency and 110% residual activity. Hyperactivation of the enzyme was also achieved by the immobilization of TGM onto dextran-oleic nanoparticles. The immobilization enzyme was stable at higher temperatures and its reuse was also possible. |
