VPLIV PARAMETROV NA SINTEZO ZAMREŽENIH ENCIMSKIH SKUPKOV (CLEAs) IZ ENCIMA CELULAZA

Petko, Maša

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Title:	VPLIV PARAMETROV NA SINTEZO ZAMREŽENIH ENCIMSKIH SKUPKOV (CLEAs) IZ ENCIMA CELULAZA
Authors:	ID Petko, Maša (Author) ID Leitgeb, Maja (Mentor) More about this mentor... ID Primožič, Mateja (Comentor)
Files:	UN_Petko_Masa_2015.pdf (1,96 MB) MD5: C013987903ED46E2FF384BA287BD45ED
Language:	Slovenian
Work type:	Undergraduate thesis
Typology:	2.11 - Undergraduate Thesis
Organization:	FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:	V diplomskem delu je opisana sinteza encimskih skupkov ali agregatov iz encima celulaza, katere na kratko imenujemo CLEAs (Crosslinked enzyme aggregates). Zamrežene encimske skupke smo pripravili po že znanem postopku, ki je sestavljen iz dveh ključnih delov. Prvi del predstavlja oboritev encima s pomočjo obarjalnega reagenta, ki je lahko organsko topilo ali raztopina anorganske soli. Temu sledi zamreženje oborjenega encima s pomočjo zamrežitvenega reagenta. Glutaraldehid je organska molekula in se najpogosteje uporablja kot zamrežitveni reagent, predvsem zaradi enostavne uporabe in relativno nizke nabavne cene. Pri sintezi CLEAs smo optimirali parametre z namenom izboljšanja relativne aktivnosti encimskih agregatov. V fazi obarjanja smo ugotavljali, kateri so tisti obarjalni reagenti, ki nam ne povzročijo ireverzibilne denaturacije, saj s tem deaktiviramo encim in izgubimo nativno aktivnost encima. Na koncu smo določili tudi stabilnost sintetizirane CLEAs, s poskusi ponovne uporabe imobiliziranega encima. Z eksperimentalnim delom smo uspešno sintetizirali zamrežene encimske skupke iz encima celulaze. Sintezo smo izvajali pri sobni temperaturi z uporabo pufra PBS (20 mM in pH = 7), s koncentracijo albumina γalbumin = 17 mg/mL in z Vrazt. celulaze = 100 µL. Za obarjanje smo uporabili 90 % delež topil, ki smo mu smo dodali 10 % delež encima celulaze. Uporabljeni obarjalni reagenti so: metanol, etanol, propanol, 2-propanol, aceton, amonijev sulfat, tetrahidrofuran, dimetilsulfoksid, polietilen glikol (PEG 1500, 6000, 20000), kloroform, n-heptan, heksan, dimetileter, butanol, acetonitril in dekanol. V stopnji zamreženja smo uporabili tiste obarjalne reagente, kjer je bilo obarjanje uspešno. Zamreženje je potekalo 3 ure. Ugotovili smo, da je bila najvišja aktivnost CLEAs pri uporabi obarjalnega reagenta etanola in pri koncentraciji glutaraldehida ϕ = 0,0625 %. Končni produkt sinteze CLEAs je bila motna suspenzija, kjer je bila povprečna velikost agregatov 43 µm. Kljub visoki aktivnosti smo ugotovili, da CLEAs ni stabilna, saj dobimo razpolovno dobo encima že po 2. ciklu. V začetni fazi raziskave smo sintetizirali CLEAs s sicer nižjo aktivnostjo, vendar so bili dobljeni agregati stabilnejši. S tem smo ovrgli trditev, da je aktivnejša CLEAs tudi stabilnejša.
Keywords:	zamreženi encimski skupki, CLEAs, celulaza, mrežni povezovalec, glutaraldehid, obarjalni reagenti
Place of publishing:	Maribor
Publisher:	[M. Petko]
Year of publishing:	2015
PID:	20.500.12556/DKUM-54840
UDC:	577.151.5(043.2)
COBISS.SI-ID:	19429654
NUK URN:	URN:SI:UM:DK:LWN9FIHS
Publication date in DKUM:	21.10.2015
Views:	2275
Downloads:	166
Metadata:
Categories:	KTFMB - FKKT
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Secondary language

Language:	English
Title:	THE INFLUENCE OF THE PARAMETERS ON THE SYNTHESIS CROSS - LINKED ENZYME AGGREGATES (CLEAS) OF ENZYME CELLULASE
Abstract:	In this work we described the synthesis of cross-linked aggregates of enzyme cellulase; shortly CLEAs. We prepared cross-linked aggregates within two major steps: precipitation and cross-linking. The first step is precipitation. As precipitating reagents we can use organic solvents or solutions of an inorganic salts. The second step is cross-linking with cross-linking reagent, which is usually glutaraldehyde. Glutaraldehyde is an organic molecule that is easy to work with and it is relatively cheap. We changed parameters in synthesis of cross-linked enzyme aggregates in the way of getting better results; higher activity of cross-linked enzyme aggregates. In precipitation phase, we needed to find precipitating reagents, which didn't cause irreversible reaction. The aim of this work is to find out how stable is synthesized CLEAs, because stability is one of the most important advantage of immobilized enzyme. We successfully synthesized CLEAs with enzyme cellulase. The parameters of synthesis were: room temperature, albumin concentration γ=17 mg/mL, volume of enzyme solutio V= 100 µL and PBS buffer (20 mM, pH=7). As precipitating reagents we used many solvents and in the phase of cross-linking we used those reagents with successfully precipitation. Cross-linking of enzyme took 3 hours. We synthesized CLEAs with the highest activity with ethanol as solvent and gluteraldehyde concentration ϕ= 0,0625 %. As result we got cloudy suspension with 43 µm of average size of the aggregates. Despite high activity we showed that CLEAs wasn't stable, because we got enzyme half-life after second cycle. During our research we proved that CLEAs with the highest activity isn't necessary also the most stable, because we synthesized CLEAs with lower activity and it was more stable than CLEAs with the highest activity.
Keywords:	cross-linked aggregates, CLEAs, cellulase, cross-linking reagent, glutaraldehyde, precipitating reagents

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