diplomsko delo univerzitetnega študijskega programa I. stopnje
Sara Štravs (Author), Maja Leitgeb (Mentor), Katja Heržič (Co-mentor)

Abstract

Encimi, v največjem deležu proteini, so znani tudi kot biološki katalizatorji. Pospešujejo kemijske reakcije v živih organizmih in so ključni biokatalizatorji z obsežnimi aplikacijami v biotehnologiji. Njihovo uporabo pogosto omejuje nizka stabilnost in možnost enkratne uporabe. Postopek imobilizacije izboljšuje stabilnost encima in omogoča, da encim večkrat uporabimo. Diplomsko delo se osredotoča na optimiranje pogojev imobilizacije lizocima na magnetne nanodelce, prevleče z arabinogalaktanom, z namenom, da bi dosegli najvišjo aktivnost imobiliziranega encima in učinkovitost imobilizacije. Arabinogalaktan (AG) je biopolimer, sestavljen iz monosaharidov arabinoze in galaktoze [1]. Najprej smo izvedli sintezo magnetnih nanodelcev, prevlečenih z arabinogalaktanom (AG-MNPs). Tako sintetizirane in modificirane magnetne nanodelce smo uporabili kot nosilec za imobilizacijo encima lizocima. Pred imobilizacijo lizocima, smo delce funkcionalizirali z mrežnim povezovalcem gluteraldehidom (GA). Uporaba GA pomembno vpliva na aktivnost encima in učinkovitost imobilizacije, saj zagotavlja proste funkcionalne skupine, na katere se lahko veže encim. Določili smo optimalno koncentracijo encima, rotacijsko hitrost stresanja med funkcionalizacijo in imobilizacijo (350 obr/min), čas funkcionalizacije zamreževalca na magnetne nanodelce (1 ura) in čas imobilizacije lizocima (2 uri) ter koncentracijo dodanega GA kot zamreževalca. Aktivnost imobiliziranega encima in učinkovitost imobilizacije smo določili z aktivnostnim testom za lizocim ter Bradfordovo metodo za določevanje totalnih proteinov. Rezultati so pokazali, da je lizocim koncentracije 30 mg/mL, ki ga imobiliziramo na predhodno funkcionalizirane delce z GA koncentracije 20 (v/v) %, imel najvišjo aktivnost (148 %) Optimirali smo čas funkcionalizacije (1 ura) in imobilizacije (2 uri) pri hitrosti stresanja 350 obratov/min. V primeru imobilizacije lizocima brez zamreževalca, smo optimirali rotacijsko hitrost 350 obr/min, koncentracijo encima 20 mg/mL in čas imobilizacije, ki znaša 2 uri.

Keywords

lizocim;imobilizacija encima;AG-MNPs;preostala aktivnost;diplomske naloge;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher: [S. Štravs]
UDC: 577.151(043.2)
COBISS: 212290307 Link will open in a new window
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Downloads: 8
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Other data

Secondary language: English
Secondary title: Enzyme immobilization on arabinogalactan-coated magnetic nanoparticles (MNPs)
Secondary abstract: Enzymes, primarily proteins, are also known as biological catalysts. They accelerate chemical reactions in living organisms and are key biocatalysts with extensive applications in biotechnology. However, their use is often limited by low stability and the possibility of single-use application. The immobilization process enhances enzyme stability and allows for their recyclability. This thesis focuses on optimizing the conditions for the immobilization of lysozyme onto magnetic nanoparticles coated with arabinogalactan, aiming to achieve the highest enzyme activity and immobilization efficiency. Initially, we synthesized magnetic nanoparticles coated with arabinogalactan (AG-MNPs). These synthesized and modified magnetic nanoparticles were then used as a carrier for the immobilization of lysozyme. Before the immobilization process, the particles were functionalized with the cross-linking agent glutaraldehyde (GA). The use of GA significantly impacts enzyme activity and immobilization efficiency by adding free functional groups to the surface of nanoparticles, which can then bind to the enzyme. Optimal enzyme concentration, rotational speed, functionalization and immobilization time, and the concentration of added GA as a cross-linking agent were determined. The activity of the immobilized enzyme and the efficiency of immobilization were determined using a lysozyme activity assay and the Bradford method for total protein determination. The results showed that lysozyme with a concentration of 30 mg/mL, cross-linked with glutaraldehyde (20 (v/v) %), exhibited the highest activity (148 %). We optimized the functionalization time (1 hour) and immobilization time (2 hours) at 350 rpm. In the case of enzyme immobilization without a cross-linker, we optimized the rotational speed to 350 rpm, enzyme concentration to 20 mg/mL, and immobilization time to 2 hours.
Secondary keywords: lysozyme;enzyme immobilization;AG-MNPs;enzyme activity;
Type (COBISS): Bachelor thesis/paper
Thesis comment: Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages: 1 spletni vir (1 datoteka PDF (VIII, 27 f.))
ID: 24815620