magistrsko delo
Tina Oprešnik (Author), Maja Leitgeb (Mentor), Katja Heržič (Co-mentor)

Abstract

Namen magistrske naloge je bil sintetizirati magnetne nanodelce s koprecipitacijo železovih oksidov, jih prevleči z CM-dekstranom ter na njih preučiti koimobilizacijo encima ADH ter kofaktorja NAD+. Po aktivaciji nanonosilcev z mrežnim povezovalcem EClH smo na njih koimobilizirali ADH in NAD+. Prostemu in imobiliziranemu encimu smo določali aktivnost z encimskim aktivnostnim testom ter izračunali preostalo aktivnost encima. Bradfordova metoda nam je služila za določanje koncentracije proteinov, s pomočjo katere smo imobilizirani ADH izračunali učinkovitost imobilizacije. Postopek koimobilizacije smo optimizirali s spreminjanjem imobilizacijskih pogojev, in sicer mase magnetnih nanodelcev, izbire mrežnega povezovalca (EClH ali GA), časa aktivacije magnetnih nanodelcev, zaporedja, časa in temperature koimobilizacije, koncentracije kofaktorja in koncentracije encima. Po določitvi optimalnih pogojev smo raziskali termično stabilnost prostega in imobiliziranega encima ter možnost skladiščenja imobiliziranega encima. Eksperimentalno smo Michaelisovi konstanti določili (KM) prosti in imobilizirani ADH. Pri optimalnih imobilizacijskih pogojih (50 mg magnetnih nanodelcev, 2 h aktivacije nanonosilcev z EClH, 2 h imobilizacije NAD+ s koncentracijo 20 mM, 4 h imobilizacije ADH s koncentracijo 0,01 mg/mL, sobna temperatura, hitrost stresanja 350 obr/min) smo dosegli najvišjo 73 % preostalo aktivnost ADH in 94 % učinkovitost imobilizacije. Imobiliziran encim je bil v primerjavi s prostim encimom termično stabilnejši, prav tako ga je bilo mogoče skladiščiti pri 4 °C. Eksperimentalno določena KM je bila pri imobilizirani ADH višja.

Keywords

alkohol dehidrogenaza;NAD+;koimobilizacija;magnetni nanodelci;CM-dekstran;magistrske naloge;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher: [T. Oprešnik]
UDC: 544.725(043.2)
COBISS: 22487830 Link will open in a new window
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Downloads: 63
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Other data

Secondary language: English
Secondary title: Co-Immobilization of alcohol dehydrogenase and nicotinamide adenine dinucleotide onto magnetic nanoparticles
Secondary abstract: The purpose of the master's thesis was to synthesize magnetic nanoparticles (MNPs) by coprecipitation method of ferric and ferrous ions and later on, coated with CM-dextran. Co-immobilization of the ADH enzyme and the NAD+ cofactor on CM-dextran coated MNPs was studied. After activation of nanoparticles with cross-linking agent EClH, ADH and NAD+ were co-immobilized. Activity of free and immobilized ADH was determined, using ADH activity assay, and residual activity of enzyme was calculated. The Bradford method was used to determine protein concentration and immobilization efficiency of immobilized ADH was calculated. The co-immobilization procedure was optimized by changing the immobilization conditions, which included: mass of MNPs, choice of cross-linking agents (EClH or GA), activation time of MNPs, sequence, time and temperature of co-immobilization process, concentration of cofactor and concentration of enzyme. After determining optimal conditions, thermal stability of free and immobilized ADH was investigated, Storage of immobilized ADH was studied, as well.. We experimentally determined Michaelis's constant (KM) of free and immobilized ADH. At optimal immobilization conditions (50 mg of MNPs, 2 hours activation of MNPs with EClH, 2 hours immobilization of NAD+ with a concentration of 20 mM, 4 hours immobilization of ADH with a concentration of 0.01 mg/mL, at room temperature and 350 rpm) the highest residual activity of ADH (73 %) and immobilization effciency (94 %) was achieved.. The immobilized enzyme was thermally more stable compared to the free enzyme, and could also be stored at 4 °C. The experimentally determined KM of immobilized ADH was higher than of free ADH.
Secondary keywords: alcohol dehydrogenase;NAD+;co-immobilization;MNPs;CM-dextran;
URN: URN:SI:UM:DK:R6NKRZPI
Type (COBISS): Master's thesis/paper
Thesis comment: Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages: XI, 50 str.
ID: 11161518