Sinteza in karakterizacija nekaterih mikro- in nanonosilcev za imobilizacijo encimov : doktorska disertacija

slovenian

doktorska dela

2015

Univerza v Mariboru, Fakulteta za kemijo in kemijsko tehnologijo

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.

07.08.2023