Imobilizirana â-laktamaza za čiščenje odpadnih vod

diplomsko delo univerzitetnega študijskega programa I. stopnje

Tajda Senekovič (Author), Maja Leitgeb (Mentor), Mateja Primožič (Co-mentor), Katja Heržič (Co-mentor)

Abstract

Antibiotiki so vrsta protimikrobnih zdravil, ki se uporabljajo za zdravljenje bakterijskih okužb. Odkritje penicilina (PEN) je bilo nedvomno eno najpomembnejših znanstvenih odkritij v novejši zgodovini, saj je rešilo mnogo življenj. Vendar pa je kljub uspešnemu zdravljenju z antibiotiki dolgotrajna uporaba le-teh privedla do razvoja odpornosti bakterij na te zdravilne učinkovine. Ko se antibiotiki izločajo in končajo v okolju, lahko vplivajo na ekosisteme in s tem na razvoj bakterijske odpornosti. To je postal globalen izziv, ki zahteva razvoj novih pristopov k odstranjevanju antibiotikov iz okolja. S tem namenom smo v diplomski nalogi izvedli študijo razgradnje PEN z uporabo imobiliziranega encima na aminosilanske magnetne nanodelce (AMN-MNPs). Uporabili smo encim β-laktamazo, ki razgrajuje beta laktamske antibiotike, med katere spada tudi PEN. Najprej smo sintetizirali AMN-MNPs na katere smo imobilizirali encim β-laktamazo. Proučili smo termično stabilnost in večkratno uporabo imobiliziranega encima ter določili aktivnost imobiliziranega encima in učinkovitost imobilizacije. Izvedli smo tudi primerjavo encimske kinetike med prosto in imobilizirano β-laktamazo z eksperimentalno določitvijo KM in vmax. Proučevali smo razgradnjo PEN s prostim in imobiliziranim encimom ter vpliv koncentracije encima in koncentracije PEN na hitrost razgradnje le-tega. Razgradnjo PEN smo zasledovali s pomočjo HPLC sistema. Rezultati so pokazali, da je bila učinkovitost imobilizacije 100,00 %. Imobilizirano β-laktamazo lahko večkrat uporabimo in je termično stabilnejša od proste β-laktamaze. Z določitvijo kinetičnih parametrov smo ugotovili, da ima imobilizirana β-laktamaza višjo afiniteto do substrata kot prosta β-laktamaza, kar pomeni, da se imobilizirana β-laktamaza učinkovitejše veže s substratom kot prosta β-laktamaza. Pri študiji razgradnje PEN smo ugotovili, da imobiliziran encim bolj učinkovito razgrajuje PEN (koncentracije 0,1 mg/mL) kot prosti encim, saj smo po 24 urah z imobilizirano β-laktamazo dosegli 97,75 % razgradnje PEN, medtem ko smo s prosto β-laktamazo dosegli le 22,21 % razgradnje PEN. Ko smo proučevali vpliv različnih koncentracij PEN smo ugotovili, da višja kot je koncentracija PEN, počasneje poteka njegova razgradnja. PEN koncentracije 0,05 mg/mL se je po petih urah popolnoma razgradil, medtem ko smo v istem času dosegli 45,00 % delež razgradnje PEN s koncentracijo 0,5 mg/mL. Na koncu smo znižali koncentracijo PEN na 0,01 mg/mL in ugotovili smo, da je se je po petih urah razgradilo 96,11 %, s tem pa smo potrdili, da razgradnja PEN poteka najhitreje pri koncentraciji 0,05 mg/mL.

Keywords

β-laktamaza;odpadna voda;penicilin;razgradnja;imobilizacija encima;MNPs;HPLC;diplomske naloge;

Data

Language:	Slovenian
Year of publishing:	2023
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[T. Senekovič]
UDC:	628.179.2(043.2)
COBISS:	166489347
Views:	38
Downloads:	12
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	Immobilized â-lactamase for wastewater treatment
Secondary abstract:	Antibiotics are a type of antimicrobial medicine used to treat bacterial infections. The discovery of penicillin (PEN) was undoubtedly one of the most important scientific discoveries in recent history, saving many lives. However, despite successful treatment with antibiotics, their long-term use has led to the development of bacterial resistance to these medicinal agents. When antibiotics are excreted and end up in the environment, they can affect ecosystems and thus the development of bacterial resistance. This has become a global challenge that requires the development of new approaches to removing antibiotics from the environment. With this aim, we conducted a study of the degradation of PEN using an immobilized enzyme on aminosilane magnetic nanoparticles (AMN-MNPs) in the thesis. We used the β-lactamase enzyme, which breaks down beta-lactam antibiotics, including PEN. First, we synthesized AMN-MNPs on which we immobilized the β-lactamase enzyme. The thermal stability and reusability of the immobilized enzyme were studied, and the activity of the immobilized enzyme and the immobilization efficiency were determined. We also performed a comparison of enzyme kinetics between free and immobilized β-lactamase by experimentally determining KM and vmax. We studied the degradation of PEN with free and immobilized enzyme and the influence of enzyme concentration and PEN concentration on the rate of its degradation. The degradation of PEN was monitored using an HPLC system. The results showed that the immobilization efficiency was 100.00%. Immobilized β-lactamase can be used repeatedly and is thermally more stable than free β-lactamase. By determining the kinetic parameters, we found that the immobilized β-lactamase has a higher affinity to the substrate than the free β-lactamase, which means that the immobilized β-lactamase binds to the substrate more efficiently than the free β-lactamase. In the study of PEN degradation, we found that the immobilized enzyme degrades PEN more efficiently (concentrations of 0.1 mg/mL) than the free enzyme, as after 24 hours with immobilized β-lactamase we achieved 97.75% of PEN degradation, while with free β-lactamase achieved only 22.21% of PEN degradation. When we studied the influence of different PEN concentrations, we found that the higher the PEN concentration, the slower its degradation. PEN with a concentration of 0.05 mg/mL was completely degraded after five hours, while in the same time we achieved a 45.00% degradation rate for PEN with a concentration of 0.5 mg/mL. Finally, we lowered the concentration of PEN to 0.01 mg/mL and found that after five hours 96.11% was degraded, thereby confirming that the degradation of PEN occurs the fastest at a concentration of 0.05 mg/mL.
Secondary keywords:	β-lactamase;wastewater;degradation;enzyme immobilization;
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	1 spletni vir (1 datoteka PDF (XI, 48 f.))
ID:	19808527