Encimska razgradnja ciprofloksacina z uporabo zamreženih encimskih skupkov

diplomsko delo visokošolskega strokovnega študijskega programa I. stopnje

Tjaša Cmager (Author), Maja Leitgeb (Mentor), Mateja Primožič (Co-mentor), Gordana Hojnik Podrepšek (Co-mentor)

Abstract

Antibiotiki so zdravila, ki nam pomagajo pri zdravljenju mnogih bolezni z zaviranjem in preprečevanjem procesov v mikrobnih celicah. Težava se pojavi pri njihovi razgradnji, saj jih človeško telo ne presnavlja v celoti. Zato se ti sproščajo v vodno okolje, kjer lahko negativno vplivajo na ne ciljne vrste. Pri diplomskem delu smo uporabljali antibiotik Ciprinol, ki se uporablja kot raztopina za infudiranje. Glavna učinkovina zdravila je Ciprofloksacin (CIP), ki spada med fluorokinolone in ima širok spekter uporabe. S tem namenom smo izvedli študijo razgradnje CIP z uporabo imobiliziranega encima v obliki zamreženih encimskih skupkov (CLEAs) in magnetnih zamreženih encimskih skupkov (mCLEAs), s katerimi smo želeli doseči čim višjo razgradnjo antibiotika. Pri tem smo uporabili imobiliziran encim lakaza, ki je najbolj raziskan in uporabljen encim pri razgradnji farmacevtskih izdelkov. Tako smo najprej izvedli sintezo CLEAs in mCLEAs, pri čemer smo za imobilizacijo uporabili mrežni povezovalec glutaraldehid (GA). Imobilizirani lakazi v obliki CLEAs in mCLEAs smo določili aktivnost ter proučili učinkovitost imobilizacije. Diplomsko delo prikazuje študijo priprave imobiliziranega encima lakaza v obliki CLEAs in mCLEAs pri čemer smo proučili možnost večkratne uporabe imobilizirane lakaze v obliki CLEAs in mCLEAs, stabilnost imobilizirane lakaze pri 4 °C in 25 °C, ter vpliv temperature na stabilnost imobilizirane lakaze v obliki CLEAs in mCLEAs (25, 35, 40, 50 in 60 °C). Razgradnjo CIP smo določili s pomočjo tekočinske kromatografije visoke ločljivosti (HPLC). Rezultati študija stabilnosti imobilizirane lakaze pri 4 °C, kažejo, da je lakaza imobilizirana v obliki CLEAs (Lak-CLEAs) bolj stabilna, v primerjavi z lakazo imobilizirano v obliki mCLEAs (Lak-mCLEAs), saj je po 56 dnevih stabilnost Lak-CLEAs znašala 93 %, medtem ko je aktivnost Lak-mCLEAs znašala le 40 %. Hkrati smo ugotovili, da je skladiščenje imobilizirane lakaze pri 25 °C manj ugodno, saj je po 21 dneh skladiščenja pri 25 °C preostala aktivnost Lak-CLEAs znašala le 77 %, Lak-mCLEAs pa samo 48 %. Študija vpliva temperature na stabilnost imobiliziranega encima v obliki CLEAs in mCLEAs, je pokazala, da je Lak-CLEAs ohranila 86,3 % svoje začetne aktivnosti po 24 h pri 25 °C in Lak-mCLEAs 72,2 % aktivnosti po 24 h pri 35 °C. Cilj diplomskega dela je bila razgradnja antibiotika CIP, pri čemer smo po 72 h dosegli 22,8 % razgradnjo z Lak-mCLEAs in 10,7 % razgradnjo CIP na 1 mg encima z Lak-CLEAs. Ugotovili smo, da je najvišja razgradnja CIP bila dosežena pri 25 °C, z uporabo mCLEAs in mediatorjem SA (4 mM) po 72 h razgradnje (22,8 %).

Keywords

lakaza;imobilizacija;diplomske naloge;

Data

Language:	Slovenian
Year of publishing:	2022
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[T. Cmager]
UDC:	66.098:577.15(043.2)
COBISS:	132354819
Views:	80
Downloads:	17
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	Enzymatic degradation of ciprofloxacin using cross-linked enzyme aggregates
Secondary abstract:	Antibiotics are drugs that help us treat many diseases by inhibiting and preventing processes in microbial cells. The problem arises with their breakdown, since the human body does not fully metabolize them. Therefore, these are released into the aquatic environment, where they can have a negative impact on non-target species. In the thesis, we used the antibiotic Ciprinol, which is used as a solution for infusion. The main active ingredient of the drug is Ciprofloxacin (CIP), which belongs to the fluoroquinolones and has a wide range of uses. With this aim, we conducted a study of CIP degradation using immobilized enzyme in the form of cross-linked enzyme assemblies (CLEAs) and magnetic cross-linked enzyme assemblies (mCLEAs), with which we wanted to achieve the highest possible degradation of the antibiotic. We used the immobilized laccase enzyme, which is the most researched and used enzyme in the degradation of pharmaceutical products. Thus, we first performed the synthesis of CLEAs and mCLEAs, using the network linker glutaraldehyde (GA) for immobilization. The activity of immobilized laccases in the form of CLEAs and mCLEAs was determined and the efficiency of immobilization was studied. The thesis shows the study of the preparation of the immobilized laccase enzyme in the form of CLEAs and mCLEAs, where we studied the possibility of multiple use of the immobilized laccase in the form of CLEAs and mCLEAs, the stability of the immobilized laccase at 4 °C and 25 °C, and the influence of temperature on the stability of the immobilized laccase in the form of CLEAs and mCLEAs (25, 35, 40, 50 and 60 °C). The degradation of CIP was determined using high-performance liquid chromatography (HPLC). The results of the stability study of immobilized laccase at 4 °C show that laccase immobilized in the form of CLEAs (Lak-CLEAs) is more stable compared to laccase immobilized in the form of mCLEAs (Lak-mCLEAs), since after 56 days the stability of Lak-CLEAs was 93%, while the activity of Lak-mCLEAs was only 40%. At the same time, we found that the storage of immobilized laccase at 25 °C is less favorable, since after 21 days of storage at 25 °C, the remaining activity of Lak-CLEAs was only 77%, and that of Lak-mCLEAs was only 48%. A study of the effect of temperature on the stability of the immobilized enzyme in the form of CLEAs and mCLEAs showed that Lak-CLEAs retained 86.3% of their initial activity after 24 h at 25 °C and Lak-mCLEAs 72.2% of their activity after 24 h at 35 °C. The goal of the thesis was the degradation of the antibiotic CIP, whereby after 72 h we achieved 22.8% degradation with Lak-mCLEAs and 10.7% degradation of CIP per 1 mg of enzyme with Lak-CLEAs. We found that the highest CIP degradation was achieved at 25 °C, using mCLEAs and SA mediator (4 mM) after 72 h of degradation (22.8%).
Secondary keywords:	laccase;immobilization;CLEAs;mCLEAs;CIP;SA;HPLC;
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	1 spletni vir (1 datoteka PDF (X, 48 f.))
ID:	16002501