diplomsko delo visokošolskega strokovnega študijskega programa I. stopnje
Anja Zirdum (Author), Maja Leitgeb (Mentor), Mateja Primožič (Co-mentor), Nika Kučuk (Co-mentor)

Abstract

Encimi so biološke molekule, sestavljene iz enega ali več polipeptidnih verig, ki se zvijejo v specifične tridimenzionalne oblike. Njihova specifična tridimenzionalna struktura omogoča vezavo substratov na aktivna mesta, kjer se izvajajo katalitične reakcije. Specifičnost encimov izhaja iz natančnega prileganja med encimom in substratom, kar pogosto opisujemo s pojmom "ključ-ključavnica". Vsak encim je tako usmerjen k določenemu substratu ali vrsti reakcije, kar omogoča visoko stopnjo regulacije in učinkovitosti v bioloških sistemih. Veliko prednost predstavlja imobilizacija encimov na trdne nosilce, kar omogoča njihovo ponovno uporabo in izboljša stabilnost ter učinkovitost encima. Uporaba imobiliziranih encimov je zaradi lažjega ločevanja od reakcijskih zmesi in posledično nižjih stroškov procesa vse bolj zaželena v industrijskih in biotehnoloških panogah. Encim transglutaminaza (TGM) se uporablja v različnih panogah, veliko vlogo ima predvsem v medicini in živilski panogi, kjer ga pogosto uporabljajo za izboljšanje teksture mesa, mlečnih izdelkov in drugih prehrambenih izdelkov. Diplomsko delo najprej zajema proizvodnjo micelarnih struktur z gojenjem medicinske gobe Ganoderma lucidum v tekočem hranilnem mediju s sladnim in kvasnim ekstraktom pri 28 °C pri statičnih pogojih. Za tvorbo micelarnih struktur je bilo potrebno gojiti G. lucidum 14 dni. Povprečna debelina micelarnih struktur je bila 0,493 mm, povprečna masa pa 3,961 g. Proizvedene micelarne strukture so izkazale visoko sposobnost absorbiranja vode z doseženim odstotkom nabrekanja po 24 h 579,3 %. S FTIR analizo smo potrdili prisotnost proteinov, lipidov in polisaharidov, s SEM analizo pa prisotnost por in hif v micelarnih strukturah. Povprečni premer por je znašal 40 µm, povprečni premer hif pa 425 nm. V nadaljevanju študije smo izvedli stabilizacijo terapevtskega encima TGM z imobilizacijo na sintetizirane micelarne strukture z metodo adsorpcije in določili učinkovitost in zmogljivost ujetja encima. Najvišja učinkovitost imobilizacije TGM v micelarne strukture je znašala 41,98 % , kar ustreza masi vgrajenega encima 8,97 g. S študijo sproščanja smo ugotovili, da se je encim uspešno adosrbiral, saj se encim niti po 24 h ni sprostil iz micelarnih struktur. Najverjetneje je adsorbirana TGM v micelarne strukture katalizirala tvorbo kavalentnih vezi med proteini in je prišlo do zamreženja TGM v notranjosti micelarnih struktur, ki vsebuje tudi proteine. Zato smo proučili potek encimske reakcije z imobiliziranim encimom v micelarne strukture pri različnih koncentracijah substrata in kinetiko imobiliziranega encima primerjali s kinetiko prostega encima. Določili smo kinetična parametra, kot sta Michaelis-Mentenova konstanta (KM) in maksimalna reakcijska hitrost (vmax) za prostiin imobilizirani encim z Lineweaver-Burkovim diagramom. Pri tem smo ugotovili, da ima sicer imobiliziran encim slabšo afiniteto do substrata kot prosti encim, zaradi višje izračunane vrednosti KM in nižje vmax. Sledilo je proučevanje vpliva mase ujetega encima na potek encimske reakcije v odvisnosti od časa. Ugotovili smo, da z naraščanjem mase imobiliziranega encima, reakcija, torej pretvorba substrata v produkt, poteka hitreje. Pri najvišji možni ujeti masi encima (8,97 mg ) v micelarne strukture smo dosegli plato, saj ni bilo zaznati signifikantnih razlik med profilom poteka encimske reakcije med 8,97 mg (13 mg/mL) in 8,76 mg ujetega encima (10 mg/mL). Imobilizacija encima na trdni nosilec je primerna za številne aplikacije. Z uspešno imobilizacijo encima TGM v micelarne strukture medicinske gobe G. lucidum, smo pripravilli stabilen funkcionalizirani naravni biokompozit, ki se lahko uporabi za različne biomedicinske aplikacije, predvsem za celjenje ran.

Keywords

Ganoderma lucidum;micelarne strukture;encimi;transglutaminaza;imobilizacija;encimski test;diplomske naloge;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher: [A. Zirdum]
UDC: 66.02(043.2)
COBISS: 221660163 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Micellar structures for the stabilization of therapeutic enzymes
Secondary abstract: Enzymes are biological molecules composed of one or more polypeptide chains that fold into specific three-dimensional shapes. Their specific three-dimensional structure allows the binding of substrates to active sites where catalytic reactions are carried out. The specificity of the enzymes arises from the precise fit between enzyme and substrate, often described by the term 'key-lock'. Each enzyme is thus targeted to a specific substrate or reaction type, allowing a high degree of regulation and efficiency in biological systems. Immobilisation of enzymes on solid supports is a major advantage, allowing their reuse and improving the stability and efficiency of the enzyme. The use of immobilised enzymes is increasingly desirable in the industrial and biotechnological sectors due to their easier separation from reaction mixtures and the resulting lower process costs. The enzyme transglutaminase (TGM) is used in a variety of industries and plays a major role in the medical and food industries, where it is often used to improve the texture of meat, dairy and other food products. The thesis firstly covers the production of micellar structures by cultivation of the medicinal mushroom Ganoderma lucidum in a liquid nutrient medium with malt and yeast extract at 28 °C under static conditions. G. lucidum had to be grown for 14 days to form the micellar structures. The average thickness of the micellar structures was 0,493 mm and the average weight was 3,961 g. The micellar structures produced showed a high water absorption capacity, with a swelling percentage after 24 h of 579,3 %. The presence of proteins, lipids and polysaccharides was confirmed by FTIR analysis, and the presence of pores and hyphae in the micellar structures was confirmed by SEM analysis. The average pore diameter was 40 µm and the average hif diameter was 425 nm. In a further study, the stabilisation of the therapeutic enzyme TGM by immobilisation on the synthesised micellar structures was carried out by adsorption method and the efficiency and entrapment capacity of the enzyme was determined. The maximum efficiency of immobilisation of TGM into the micellar structures was 41,98 %, corresponding to a mass of incorporated enzyme of 8,97 g. The release study showed that the enzyme was successfully adsorbed, as the enzyme was not released from the micellar structures even after 24 h. Most probably, the adsorbed TGM in the micellar structures catalysed the formation of covalent bonds between proteins and crosslinking of the TGM in the interior of the micellar structures, which also contains proteins, occurred. Therefore, the course of the enzymatic reaction with the enzyme immobilised in the micellar structures was studied at different substrate concentrations and the kinetics of the immobilised enzyme was compared with the kinetics of the free enzyme. Kinetic parameters such as Michaelis-Menten constant (KM) and maximum reaction rate (vmax) for the free and immobilised enzyme were determined using a Lineweaver-Burk diagram. We found that the immobilised enzyme has a lower affinity for the substrate than the free enzyme, due to the higher calculated KM and lower vmax. The effect of the mass of the entrapped enzyme on the time-dependence of the enzymatic reaction was then studied. It was found that as the mass of immobilised enzyme increases, the reaction, i.e. the conversion of substrate to product, proceeds faster. A plateau was reached at the highest possible mass of enzyme trapped (8,97 mg ) in the micellar structures, as no significant differences were detected in the enzyme reaction profile between 8,97 mg (13 mg/mL) and 8,76 mg of trapped enzyme (10 mg/mL). Immobilisation of the enzyme on a solid support is suitable for many applications. By successfully immobilizing the TGM enzyme into the micellar structures of the medicinal mushroom G. lucidum, we have prepared a stable functionalized natural biocomposite that can be used for a variety of biomedical applicat
Secondary keywords: enzymes;transglutaminase;G. lucidum;immobilization;release kinetics;enzymes test;
Type (COBISS): Bachelor thesis/paper
Thesis comment: Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages: 1 spletni vir (1 datoteka PDF (XIII, 42 f.))
ID: 25042812