doktorska disertacija
Franja Šulek (Author), Maja Leitgeb (Mentor), Željko Knez (Co-mentor)

Abstract

V doktorski nalogi je nazorno predstavljen postopek sinteze magnetnih nanodelcev iz železovega oksida maghemita, ?-Fe2O3, za vezavo biokatalizatorja. Magnetni nanodelci, ki v zadnjih letih vse bolj pridobivajo na vrednosti kot potencialni encimski nosilci, so bili najprej sintetizirani zmetodo obarjanja ali koprecipitacije železovih (II, Fe2+) in železovih (III, Fe3+) ionov v alkalnem mediju pri hitrem mešanju in visoki temperaturi. Površinska funkcionalizacija magnetnih nanodelcev je bila izvedena v dveh stopnjah. Primarna funkcionalna prevleka iz silicijevega dioksida (SiO2), ki delcem daje predvsem višjo stabilnost, je bila sintetizirana pri striktno kontroliranih reakcijskih pogojih iz natrijevega silikata ali Na2SiO3. Sekundarni funkcionalni sloj za doseganje višje funkcionalnosti in reaktivnosti površine nanodelcev je bil sintetiziran iz organskih molekul aminosilana ali 3-(2-aminoetilamino)-propil-dimetoksimetilsilana v kislem mediju. V nadaljnjem so bili tako površinsko spremenjeni magnetni nanodelci uporabljeni za imobilizacijo specifičnega biokatalizatorja holesterol oksidaze(ChOx, EC 1.1.3.6) iz Corynebacterium sp. Analiza magnetnih nanodelcevz imobilizirano ChOx je pokazala, da so imeli nanodelci značilno obliko kroglice ali sfere s povprečnim premerom 50,2 nm. Vezava holesterol oksidaze je bila uspešno potrjena tudi s FT-IR tehniko. Učinkovitost imobilizacije na magnetni nosilec je znašala 92 % pri uporabljeni koncentraciji encima 100 g mL-1. Aktivnost imobilizirane ChOx na magnetne nanodelce, prevlečene s tanko plastjo silikatne prevleke premera 3 nm, je bila57 % v primerjavi z aktivnostjo ChOx. Študija vpliva pH-vrednosti in temperature na aktivnost in stabilnost encimskega preparata je pokazala, da ima imobilizirana ChOx višjo toleranco na spremembo pH-vrednosti okolja in višjo termično stabilnost. Prav tako je bila stabilnost imobilizirane ChOx priponovni uporabi dobra. V drugem delu doktorske disertacije je opisana priprava aktivnih encimskih skupkov iz encima peroksidaze (HRP, EC 1.11.1.7), pridobljenega iz navadnega hrena (lat.: Armoracia rusticana or Cochlearia armoracia), in postopek zamreženja le-teh z glutaraldehidom za pripravo končneoblike stabilnih zamreženih encimskih skupkov ali na kratko CLEAs. Postopek priprave CLEAs je bil razdeljen na dva ključna dela, in sicer, na obarjanje topnega ali nativnega encima s pomočjo ustreznega obarjalnega reagenta, in na nadaljnjo zamreženje tako izoborjenega encima s pomočjo mrežnega povezovalca. Končni videz CLEAs je podoben motni suspenziji, v kateriso jasno vidni skupki encimov, značilne sferične oblike in premera okrog250 nm. Uspešno smo sintetizirali zamrežene encimske skupke iz hrenove peroksidaze pod različnimi testnimi pogoji. Končna aktivnost encimskih skupkoviz HRP je bila 83 %. Zamrežene encimske skupke smo sintetizirali v prisotnosti encima albumina iz kokošjih jajc in funkcionalnega aditiva, penta-etilen-heksanamina (PEHA). Encim albumin poveča stabilnost encimskih skupkov, medtem ko PEHA poveča število prostih amino skupin (-NH2) na zunanji površini encima HRP, kar poenostavi postopek zamreženja encimskih delcev z mrežnim povezovalcem.

Keywords

biokatalizatorji;imobilizacija;encimski nosilci;nanostrukturirani materiali;magnetni nanodelci;površinska funkcionalizacija;zamreženi encimski skupki;stabilizacija biokatalizatorja;holesterol oksidaza;hrenova peroksidaza;

Data

Language: Slovenian
Year of publishing:
Source: [Maribor
Typology: 2.08 - Doctoral Dissertation
Organization: UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher: F. Šulek]
UDC: 544.473:577.15(043.3)
COBISS: 14930966 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Nanostructured materials for enzyme immobilization
Secondary abstract: The research work offers a concise guide of the synthesis of maghemite magnetic nanoparticles, ?-Fe2O3, for immobilization of a biocatalyst. Magneticnanoparticles, that are gaining an exceptionally increased attention as potential enzyme support in the recent years, were synthesized by the coprecipitation technique of ferrous (II, Fe2+) and ferric (III, Fe3+) ions inalkaline medium at harsh stirring and high temperature, respectively. Surface functionalization of magnetic nanoparticles was carried out stepwise and divided into two major steps. Primary functional layer of silica (SiO2) that enhanced the stability of magnetic nanoparticles was synthesized under strictly regulated reaction conditions from sodium silicate. Next, the secondary functional layer formed of organic molecules of amino silane or 3-(2-Aminoethylamino)-propyl-dimethoxymethylsilane in order to achieve higher functionality and reactivity of the surface of magnetic nanoparticles was synthesized in acidic reaction medium. Furthermore, the prepared magnetic nanocomposites were used for the immobilization of a specific biocatalyst cholesterol oxidase (ChOx, EC 1.1.3.6) from Corynebacterium sp. The analysis of the magnetic nanoparticles with immobilized ChOx showed that the nanoparticles adopted a typical spherical shape with a mean diameter of 50,2 nm. The binding of ChOx was successfully confirmed by FT-IR technique. The binding efficiency was 92 % and was maximally achieved at enzyme concentrationof 100 g mL-1. The activity of immobilized ChOx onto magnetic nanoparticles, coated with a thin functional layer of silica with a thickness of 3 nm, was estimated to be 57 % in comparison to its native ChOx. The effects of pH and temperature also indicated the bound ChOx had better pH-tolerance and exhibited higher thermal stability. Furthermore, the immobilized system revealed also good reusable stability. The second part of the PhD work was focused on the synthesis of active enzyme aggregates of peroxidase (HRP, EC 1.11.1.7) from horse radish roots (lat.: Armoracia rusticana or Cochlearia armoracia), further cross-linked with glutaraldehyde in order to obtain the final form of stable cross-linked enzyme aggregates or CLEAs. The procedure to prepare CLEAs was divided into two major steps, that involves first the precipitation of the soluble enzyme with a suitable precipitant such and second the crosslinking step with an appropriate cross-linker. The final suspension of CLEAs obtained was moderately turbid andenzyme particles could be normally observed with an average diameter of 250nm. The CLEAs of HRP were successfully produced under different testing conditions afterwards. The final recovery activity of the CLEAs attained was 83 % compared to the activity of native enzyme. The synthesised CLEAs were prepared in the presence of egg albumin and functional additive as pentaethylenehexamine (PEHA). The role of albumin in CLEAs preparation plays asignificant role as a stabilizing agent of CLEAs particles, whereas the addition of PEHA was fundamental to obtain fully cross-linked HRP aggregates. For, the HRP possesses only 6 Lys (lysine) amino residues, that substantially impedes the cross-linking of the enzyme to completion, the addition of PEHA increased the free amino groups (-NH2) on the outer surface of the enzyme, making the cross-linking more feasible.
Secondary keywords: immobilization of biocatalyst;enzyme supports;nanostructured materials;magnetic nanoparticles;surface functionalization;cross-linked enzyme aggregates;stabilization;cholesterol oxidase;horse radish peroxidase;Bioktaliza;Disertacije;Imobilizacija;Nanomateriali;
URN: URN:SI:UM:
Type (COBISS): Dissertation
Thesis comment: Univ. Maribor, Fak. za kemijo in kemijsko tehnologijo
Pages: XVII, 133 str., [4] f. pril.
Keywords (UDC): mathematics;natural sciences;naravoslovne vede;matematika;chemistry;crystallography;mineralogy;kemija;physical chemistry;fizikalna kemija;chemical kinetics;catalysis;mathematics;natural sciences;naravoslovne vede;matematika;biological sciences in general;biologija;material bases of life;biochemistry;molecular biology;biophysics;biokemija;molekularna biologija;biofizika;
ID: 1014117