doktorska disertacija
Gordana Hojnik Podrepšek (Author), Maja Leitgeb (Mentor), Željko Knez (Co-mentor)

Abstract

Doktorska disertacija je razdeljena na dva sklopa, v katerih smo razširili dosedanje raziskave na področju priprave magnetnih nanodelcev, njihove funkcionalizacije in imobilizacije encimov na tako pripravljene mikro- in nanonosilce. Osrednji cilj disertacije je priprava magnetnih nanodelcev z metodo obarjanja ali koprecipitacije železovih (II) in železovih (III) ionov v alkalnem mediju, funkcionaliziranih s hitozanom po treh različnih metodah; metodi mikroemulzije, suspenzijske zamreževalne tehnike in kovalentne vezave. Podrobna karakterizacija tako pripravljenih nosilcev je bila izvedena s pomočjo presevne elektronske mikroskopije, vrstične elektronske mikroskopije, rentgenske praškovne difrakcije, z dinamičnim sipanjem laserske svetlobe, laserskim granulometrom in merjenjem specifične magnetizacije. Tako smo opazovali morfologijo in lastnosti magnetnih nosilcev. Površinsko funkcionalizacijo s hitozanom za doseganje višje funkcionalnosti smo potrdili s pomočjo energijskodisperzijske spektroskopije, termične analize, potenciometrične titracije in s Fourierevo transformacijo. Z mikrobiološkim testiranjem je bil določen vpliv magnetnih mikro- in nanonosilcev na rast mikrobnih kultur. Nadalje smo magnetne mikro- in nanodelce, prevlečene s plastjo hitozana, uporabili kot nosilce za imobilizacijo holesterol oksidaze (EC 1.1.3.6). S spreminjanjem različnih pogojev, kot so koncentracija encima, koncentracija in vrsta mrežnega povezovalca glutaraldehida ali pentaetilen heksamina ter hitrost stresanja, smo ugotovili, kako se spreminja učinkovitost imobilizacije encima ter kolikšen delež aktivnosti se pri tem ohrani v primerjavi s prostim encimom. Aktivnost imobilizirane holesterol oksidaze smo določili z reakcijo oksidacije holesterola. Optimalni reakcijski pogoji imobilizacije holesterol oksidaze na magnetne mikro- in nanonosilce so bili doseženi pri koncentraciji encima 1 mg/mL ob uporabi 0,02 M pentaetilenheksamina kot mrežnega povezovalca. Aktivnost imobilizirane holesterol oksidaze pri optimalnih reakcijskih pogojih je bila 79,03 %. Prav tako je bila stabilnost imobilizirane holesterol oksidaze pri ponovni uporabi dobra. V drugem sklopu je opisana priprava zamreženih encimskih skupkov iz encima celulaze (EC 3.2.1.4). Postopek priprave zamreženih encimskih skupkov vključuje dva pomembna koraka, in sicer obarjanje encima z dodatkom obarjalnega reagenta in zamreženje encimskih molekul z glutaraldehidom. Optimalni reakcijski pogoji sinteze zamreženih encimskih skupkov so bili doseženi pri koncentraciji glutaraldehida 0,0625 % (v/v) in uporabi etanola kot obarjalnega reagenta. Aktivnost imobilizirane celulaze v obliki zamreženih encimskih skupkov smo določili z reakcijo hidrolize celuloze. Aktivnost imobilizirane celulaze v obliki zamreženih encimskih skupkov pri optimalnih reakcijskih pogojih je bila 93,95 %. Prav tako je bila stabilnost imobilizirane celulaze pri ponovni uporabi dobra.

Keywords

imobilizacija encimov;metoda CLEAs;hitozan;holesterol oksidaza;celulaza;zamreženi encimski skupki;Nanotehnologija;Disertacije;Magnetni nanodelci;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher: [G. Hojnik Podrepšek]
UDC: 543.645.4:577.15(043.3)
COBISS: 18931222 Link will open in a new window
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Other data

Secondary language: English
Secondary title: THE SYNTHESIS AND CHARACTERIZATION OF MICRO- AND NANOCARRIERS FOR ENZYME IMMOBILIZATION
Secondary abstract: The PhD thesis is divided in two parts, in which we enhanced existing research in the filed of preparation of magnetic nanoparticles and their functionalization and enzyme immobilization on micro- and nanocarriers. The main objective of the thesis is the preparation of magnetic nanoparticles by precipitation method of iron (II) and iron (III) ions in an alkaline medium and the functionalization with chitosan by three different methods; microemulsion, suspension cross-linking technique and covalent binding. Detailed characterization of these carriers was performed using transmission electron microscopy, scanning electron microscopy, X-ray powder diffraction, with dynamic laser light scattering, laser granulometry and the measurement of specific magnetization. Thus, we observe the morphology and the characteristics of magnetic carriers. Surface functionalization of the chitosan in order to achieve higher functionality was confirmed by energy dispersive spectroscopy, thermal analysis, potentiometric titration and Fourier transformation. The microbiological testing determined the influence of magnetic micro- and nanocarriers on the growth of microbial cultures. Furthermore, the magnetic nanoparticles coated with chitosan were used as a carriers for the immobilization of cholesterol oxidase (EC 1.1.3.6). By changing various conditions such as the enzyme concentration, concentration and type of cross-linker glutaraldehyde or pentaetylenehexamine and shaking rate, we determined an immobilization efficiency and retained activity of immobilized enzyme compared to free enzyme. The activity of the immobilized cholesterol oxidase was determined by reaction of the cholesterol oxidation. The optimal reaction conditions of cholesterol oxidase immobilization to the magnetic micro- and nanocarriers were obtained using 1mg/mL of the enzyme concentration and 0,02 M pentaetilenhexamine, as a cross-linker. Activity of the immobilized cholesterol oxidase under optimal conditions was 79,03 %. Also, the stability of immobilized cholesterol oxidase revealed good reusability. The second part describes the preparation of cross-linked enzyme aggregates of cellulase (EC 3.2.1.4). The preparation process of cross-linked enzyme aggregates includes two important steps, such as the precipitation of the enzyme by the addition of the precipitation reagent and the cross-linking of enzyme molecules with glutaraldehyde. The optimum reaction conditions of cross-linked enzyme aggregates of cellulase have been achieved at a 0,0625 % concentration of glutaraldehyde (v/v) and using ethanol as a precipitation reagent. The activity of immoblized cellulase as cross-linked enzyme aggregates was determined by reaction of hydrolysis of cellulose. Activity of the immobilized cellulase as cross-linked enzyme aggregates under optimal conditions was 93,95 %. Also, the stability of immobilized cellulase revealed good reusability.
Secondary keywords: magnetic nanoparticles;enzyme immobilization;chitosan;holesterol oxidase;cellulase;cross-linked enzyme aggregates;Encimi;Disertacije;Imobilizacija;Nanodelci;
URN: URN:SI:UM:
Type (COBISS): Doctoral dissertation
Thesis comment: Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages: XIV, 116 str.
ID: 8752663