Vezava encimov na površinsko modificirane magnetne nosilce : doktorska disertacija

Vasić, Katja

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Naslov:	Vezava encimov na površinsko modificirane magnetne nosilce : doktorska disertacija
Avtorji:	ID Vasić, Katja (Avtor) ID Leitgeb, Maja (Mentor) Več o mentorju... ID Primožič, Mateja (Komentor)
Datoteke:	DOK_Vasic_Katja_2020.pdf (9,26 MB) MD5: A46663CAA3AB350CEDD9510334246451 PID: 20.500.12556/dkum/938ac6a7-5db3-4083-99e2-2da49f8ba760
Jezik:	Slovenski jezik
Vrsta gradiva:	Doktorsko delo/naloga
Tipologija:	2.08 - Doktorska disertacija
Organizacija:	FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Opis:	Doktorska disertacija zajema dva dela, v prvem delu smo se osredotočili na sintezo magnetnega nosilca, modificiranega z organskim polimerom karboksimetil dekstranom (CMD). V sintezni postopek smo vpeljali tri različne koncentracije CMD (0,25 g/mL, 0,40 g/mL in 0,50 g/mL CMD) ter sintetizirali tri različne modificirane magnetne nanodelce (CMD1-MNPs, CMD2-MNPs in CMD3-MNPs). Sintetizirane CMD-MNPs smo okarakterizirali z različnimi analiznimi metodami: Fourier-transformirano infrardečo spektroskopijo (FT-IR), termogravimetrično analizo (TGA), vrstično elektronsko mikroskopijo (SEM), energijsko disperzijsko spektroskopijo (EDS), transmisijsko elektronsko mikroskopijo (TEM), z meritvami dinamičnega sipanja svetlobe (DLS). Magnetne lastnosti smo določili z elektronsko paramagnetno resonanco (EPR) in vibracijskim magnetometrom (VSM). Uspešno smo sintetizirali magnetne nosilce CMD-MNPs z ozkimi porazdelitvami nanovelikosti od 27-30 nm. S FT-IR analizo smo določili prisotnost karboksilnih in hidroksilnih skupin na površini CMD-MNPs, kar potrjuje prisotnost polimerne prevleke CMD na sintetiziranih MNPs. Z meritvami EPR in VSM smo dokazali, da imajo sintetizirani CMD-MNPs magnetne lastnosti ter feromagnetni sistem. Določili smo inhibitorne lastnosti CMD-MNPs na rast dveh bakterijskih kultur. Inhibitorne učinke na rast testnih mikroorganizmov smo zaznali pri CMD3-MNPs, medtem ko ostali CMD-MNPs in neprevlečeni MNPs ne izkazujejo antimikrobne učinkovitosti. Proučevali smo tudi inhibitorne lastnosti MNPs modificiranih s hitozanom (HIT-MNPs) in aminosilanom (AMS-MNPs) na petih različnih bakterijskih kulturah, pri katerih nismo zaznali inhibiornega učinka na rast izbranih mikororganizmov. Za nadaljnje raziskave smo izbrali CMD3-MNPs, na katerega smo vezali encim alkohol dehidrogenazo (ADH). V drugem delu doktorske disertacije smo nosilec CMD3-MNPs površinsko funkcionalizirali z epiklorohidrinom (EClH). Optimalna koncentracija EClH je znašala 4 % (v/v). Proučevali smo vpliv različnih procesnih parametrov na preostalo aktivnost in učinkovitost imobilizacije ADH na CMD3-MNPs. Pri optimalnih pogojih imobilizacije ADH na CMD3-MNPs smo dosegli 89,6 % preostalo aktivnost imobilizirane ADH in 99,5 % učinkovitost imobilizacije. Nadaljevali smo s postopkom koimobilizacije, pri čemer smo na funkcionaliziran nosilec CMD3-MNPs koimobilizirali encim ADH in kofaktor β-nikotinamid adenin dinukleotid (β-NAD). S spreminjanjem procesnih parametrov smo proučevali njihov vpliv na preostalo aktivnost imobilizirane ADH s kofaktorjem β-NAD na CMD3-MNPs in učinkovitost koimobilizacije. Pri optimalnih pogojih koimobilizacije ADH in β-NAD na CMD3-MNPs smo dosegli 73,3 % preostalo aktivnost ADH ter 93,8 % učinkovitost imobilizacije. Izvedli smo še študijo termične stabilnosti proste ADH, ADH imobilizirane na CMD3-MNPs in ADH koimobilizirane z β-NAD na CMD3-MNPs pri različnih temperaturah. ADH imobilizirana na CMD3-MNPs je ohranila skoraj 60 % svoje začetne aktivnosti po 24. urah inkubacije pri temperaturah 20 °C in 40 °C. ADH koimobilizirana z β-NAD na CMD3-MNPs je pri temperaturi 30 °C ohranila 75,4 % začetne aktivnosti, pri 50 °C pa 66,5 % začetne aktivnosti po 5. urah inkubacije. Proučili smo še stabilnost ADH imobilizirane na CMD3-MNPs in ADH koimobilizirane z β-NAD na CMD3-MNPs, ki smo ju skladiščili pri 4 °C. Po treh tednih sta obe obliki imobilizrane ADH ohranili 60 % svoje začetne aktivnosti. Magnetne nosilce HIT-MNPs in AMS-MNPs smo funkcionalizirali z mrežnim povezovalcem glutaraldehidom (GA) in amino-donorjem pentaetilenheksaminom (PEHA), na katere smo imobilizirali encim β-galaktozidazo (β-GAL) ter optimirali koncentraciji GA in PEHA. Pri kombinaciji obeh GA in PEHA smo dosegli hiperaktivacijo encima (128,9 %), do katere pride zaradi konformacijskih sprememb encima. Hiperaktivacijo smo dosegli tudi pri imobilizaciji β-GAL na AMS-MNPs. Najvišjo preostalo aktivnost β-GAL smo dosegli, kadar smo kot mrežni povezovalec uporabili 20 % (v/v) PEHA (154,4 %).
Ključne besede:	magnetni nanodelci, karboksimetil dekstran, karakterizacijska analiza, funkcionalizacija, epoksi zamreževanje, epiklorohidrin, glutaraldehid, encimi, imobilizacija, koimobilizacija, β-nikotinamid adenin dinukleotid, alkohol dehidrogenaza, β-galaktozidaza, kinetični parametri
Kraj izida:	Maribor
Kraj izvedbe:	Maribor
Založnik:	[K. Vasić]
Leto izida:	2020
Št. strani:	XXIV, 161 str.
PID:	20.500.12556/DKUM-75460
UDK:	543.645.4(043.3)
COBISS.SI-ID:	15207939
NUK URN:	URN:SI:UM:DK:IUW3ANIY
Datum objave v DKUM:	13.05.2020
Število ogledov:	2276
Število prenosov:	356
Metapodatki:
Področja:	KTFMB - FKKT
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Licence

Licenca:	CC BY-NC-ND 4.0, Creative Commons Priznanje avtorstva-Nekomercialno-Brez predelav 4.0 Mednarodna

Povezava:	http://creativecommons.org/licenses/by-nc-nd/4.0/deed.sl
Opis:	Najbolj omejujoča licenca Creative Commons. Uporabniki lahko prenesejo in delijo delo v nekomercialne namene in ga ne smejo uporabiti za nobene druge namene.
Začetek licenciranja:	19.11.2019

Sekundarni jezik

Jezik:	Angleški jezik
Naslov:	Enzyme immobilization on surface modified magnetic carriers
Opis:	Doctoral thesis consists of two parts, in the first part synthesis of magnetic carrier and modification with organic polymer carboxymethyl dextran (CMD) was performed. Three different CMD concentrations were applied into the synthesis process (0,25 g/mL, 0,40 g/mL in 0,50 g/mL CMD), which resulted in three different modified magnetic carriers (CMD1-MNPs, CMD2-MNPs and CMD3-MNPs). All CMD-MNPs were characterized with various analytical methods: Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDX), transmission electron microscopy (TEM), dynamic light scattering (DLS). Magnetic properties were analyzed with electron paramagnetic resonance (EPR) and magnetization measurements with vibrating sample magnetization (VSM). CMD-MNPs were successfully synthesized with average diameters from 27-30 nm and size distribution revealed most consistent sizes of CMD3-MNPs. Hydroxyl and carboxyl groups were confirmed on the CMD-MNPs surface, which confirms the presence of CMD layer on the surface of CMD-MNPs. EPR and VSM measurements confirmed magnetic properties of all CMD-MNPs and a ferromagnetic system. Inhibition properties were determined of all CMD-MNPs on two different bacterial cultures, where CMD3-MNPs displayed inhibition zone, confirming antimicrobial properties, whereas with other CMD-MNPs no inhibition was detected. Toxicity was also determined on MNPs coated with chitosan (HIT-MNPs) and aminosilane (AMS-MNPs) on five different bacterial cultures, where no inhibition was detected. CMD3-MNPs was chosen for further investigation of enzyme immobilization in the second part of doctoral thesis, because of its most favourable properties. In second part of doctoral thesis nano-carrier CMD3-MNPs was surface functionalized with epoxy cross-linking, using epoxy cross-linker epichlorohydrin (EClH) to covalently bind enzyme alcohol dehydrogenase (ADH). With optimization of process parameters EClH with 4 % (v/v) was used, which resulted in the highest residual activity of immobilized ADH. Epoxy-functionalized CMD3-MNPs were used in immobilization protocol, where effect of process parameters was investigated on the residual activity and immobilization efficiency of ADH. After successful optimization ADH immobilized onto CMD3-MNPs managed to obtain 89,6 % of residual activity and 99,5 % of immobilization efficiency. Further on, co-immobilization of enzyme ADH and cofactor β-nicotinamide adenine dinucleotide (β-NAD) was performed. Again, effect of process parameters on residual activity and immobilization efficiency of co-immobilization were investigated. After successful optimization, ADH co-immobilized with β-NAD onto CMD3-MNPs managed to obtain 73,3 % of residual activity and 93,8 % of immobilization efficiency. Thermal stability of immobilized ADH at different temperatures was investigated. ADH immobilized onto CMD3-MNPs obtained almost 60 % of its initial activity after 24 hours at 20 °C and 40 °C, ADH co-immobilized with β-NAD onto CMD3-MNPs managed to obtain 75,4 % of its initial activity at 30 °C and 66,5 % of its initial activity at 50 °C after 5 hours. Storage stability of ADH immobilized onto CMD3-MNPs and ADH co-immobilized with β-NAD onto CMD3-MNPs were investigated at 4 °C, where both obtained almost 60 % of its initial activity after three weeks. Magnetic carriers HIT-MNPs and AMS-MNPs were functionalized with cross-linker glutaraldehyde (GA) and amino-donor pentaethylenehexamine (PEHA). Functionalized HIT-MNPs and AMS-MNPs were immobilized with enzyme β-galactosidase (β-GAL). When using combination of 0,5 % (v/v) GA and 30 % (v/v) PEHA 128,9 % of residual activity was achieved, which resulted in hyper-activation of enzyme due to its conformational changes. Hyper-activation was achieved also with immobilizing β-GAL onto AMS-MNPs and the highest residual activity was obtained with with 20 % (v/v) of PEHA (154,4 %).
Ključne besede:	magnetic nanoparticles, modification, carboxymethyl dextran, characterization analysis, functionalization, epoxy cross-linking, epichlorohydrin, glutaraldehyde, enzyme, immobilization, co-immobilization, β-nicotinamide adenine dinucleotide, alcohol dehydrogenase, β-galactosidase, kinetic parameters

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