Encimska razgradnja antibiotika z uporabo imobilizirane lakaze

diplomsko delo univerzitetnega študijskega programa I. stopnje

Luna Petrovič (Author), Maja Leitgeb (Mentor), Mateja Primožič (Co-mentor), Gordana Hojnik Podrepšek (Co-mentor)

Abstract

Namen diplomskega dela je bil izvedba študije učinkovitosti razgradnje antibiotika ciprofloksacina (CIP) s pomočjo imobiliziranega encima lakaze na magnetne nanodelce (MNPs). V prvem delu smo s koprecipitacijo Fe2+ in Fe3+ ionov pripravili funkcionalizirane MNPs, ki smo jih na začetku prevlekli s plastjo citronske kisline, da smo s tem preprečili aglomeracijo delcev. Za tem smo jih prevlekli še z natrijevim silikatom in funkcionalizirali z aminosilanom, s čimer smo omogočili nadaljnjo vezavo encima. V drugem delu smo MNPs aktivirali z zamreževalnim reagentom glutaraldehidom (GA) in nanje imobilizirali lakazo. Z imobilizacijo smo encimu povečali stabilnost, kar je omogočilo tudi njegovo večkratno uporabo. Nadalje smo proučevali učinkovitost imobilizacije lakaze na visoko funkcionalizirane MNPs ter aktivnost imobiliziranega encima, pri čemer smo optimirali koncentracijo dodanega encima, čas imobilizacije, čas aktivacije z GA in volumen dodanega GA. S tako imobiliziranim aktivnim encimom smo nato izvedli reakcijo razgradnje CIP-a. Za namen boljše razgradnje CIP-a smo imobilizirani lakazi dodali mediator siringaldehid (SA), ki v veliki meri pripomore k povišanju aktivnosti lakaze. Prav tako smo proučili še vpliv temperature na stabilnost imobilizirane lakaze na MNPs ter določili kinetiko reakcije, pri čemer smo spreminjali koncentracijo substrata (CIP). Na pripravljene MNPs nam je uspelo imobilizirati več kot 95 % encima in pri tem ohraniti 77 % njegove aktivnosti. Z imobiliziranim encimom smo po 24 h pri 25 °C dosegli 88,3 %, pri 30 °C 82,9 %, pri 40 °C pa 75,0 % razgradnjo CIP-a v raztopini s koncentracijo CIP-a 30 mg/L. Ugotovili smo, da se z višanjem temperature razgradnja CIP-a slabša. Postopek razgradnje CIP-a smo izvedli tudi pri treh različnih koncentracijah raztopine CIP-a, pripravljene s fosfatnim pufrom, in sicer 0,01 mg/mL, 0,03 mg/mL ter 0,05 mg/mL. Pri višji koncentraciji substrata smo zaznali višjo stopnjo razgradnje CIP-a. Določili smo tudi K_M in v_max za imobilizirano in prosto lakazo. K_M za imobilizirano lakazo je znašal 44 mg/L, v_max pa 0,11 mg/L min, medtem ko je K_M za prosto lakazo znašal 150 mg/L, v_max pa 0,98 mg/L min.

Keywords

lakaza;MNPs;imobilizacija encima;aktivnost encima;glutaraldehid;ciprofloksacin;siringaldehid;razgradnja;diplomske naloge;

Data

Language:	Slovenian
Year of publishing:	2022
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[L. Petrovič]
UDC:	66.098:577.15(043.2)
COBISS:	121405187
Views:	11
Downloads:	4
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Enzymatic degradation of antibiotic using immobilized laccase
Secondary abstract:	The goal of the thesis was to perform a study of the effectiveness of the degradation of the antibiotic ciprofloxacin (CIP) with enzyme laccase immobilized on magnetic nanoparticles (MNPs). In the first stage, functionalized MNPs were prepared by co-precipitation of Fe2+ and Fe3+ ions, which were initially coated with a layer of citric acid to prevent particle agglomeration. After that, they were coated with sodium silicate and functionalized with aminosilane, which enabled further binding of the enzyme. In the second stage, MNPs were activated with the crosslinking reagent glutaraldehyde (GA) and laccase was immobilized onto them. By immobilizing the enzyme, we increased its stability, which enabled it to be used multiple times. We further studied the efficiency of immobilization onto highly functionalized MNPs and the activity of the immobilized enzyme using a UV-Vis spectrophotometer. Furthermore, the concentration of added enzyme, immobilization time, the time of activation with GA, and the concentration of added GA was optimized. With the immobilized active enzyme, we performed a degradation reaction of the antibiotic ciprofloxacin (CIP) and examined its efficiency with High performance liquid chromatography (HPLC) system. For better degradation of CIP, the mediator syringaldehyde (SA) was added to the immobilized laccase, which helped increase the laccase activity. The CIP solution with immobilized laccase onto the MNPs was then exposed to three different temperatures and three different concentrations of the CIP solution and their influence on the CIP degradation was observed. We managed to immobilize more than 95 % of the added enzyme onto the prepared MNPs while retaining 77 % of its activity. After 24 h of exposure with the CIP solution in a concentration of 30 mg/L, we observed a degradation rate of 88,3 % at 25 °C, 82,9 % at 30 °C, and 77,0 % at 40 °C, which shows that, with increasing temperature, the degradation efficiency of CIP deteriorates. The CIP degradation process was also carried out at three different concentrations of the CIP solution prepared with phosphate buffer, namely 0.01 mg/mL, 0.03 mg/mL and 0.05 mg/mL. A higher level of CIP degradation was detected at a higher substrate concentration. We also determined K_M and v_max for immobilized and free laccase. The K_M for immobilized laccase was 44 mg/L and v_max was 0.11 mg/L min, while the K_M for free laccase was 150 mg/L and v_max was 0.98 mg/L min.
Secondary keywords:	laccase;MNPs;enzyme immobilization;enzyme activity;glutaraldehyde;ciprofloxacin;syringaldehyde;degradation;HPLC;
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	1 spletni vir (1 datoteka PDF (XIII, 59 f.))
ID:	15950726