Sinteza zamreženih encimskih skupkov iz encima transglutaminaze

diplomsko delo univerzitetnega študijskega programa I. stopnje

Špela Lesičar (Author), Maja Leitgeb (Mentor), Katja Heržič (Co-mentor)

Abstract

Namen diplomske naloge je bila uspešna sinteza zamreženih encimskih skupkov (CLEAs) iz encima transglutaminaze. Pri tem smo želeli doseči čim višjo učinkovitost imobilizacije in preostalo aktivnost encima transglutaminaze. Sinteza je zajemala postopek obarjanja encima v izbranem obarjalnem reagentu in zamreženja encima z mrežnim povezovalcem glutaraldehidom (GA). Da bi dosegli čim boljše rezultate smo morali izbrati ustrezen obarjalni reagent ter njegovo količino. Optimirali smo parametre pri zamreženju, kot so koncentracija dodanega mrežnega povezovalca, koncentracija dodanega natrijevega cianoborohidrida (NaBH3CN), koncentracija ogrodnih proteinov govejega serumskega albumina (BSA) in jajčnega albumina (EA) ter koncentracija transglutaminaze. Iz rezultatov smo ugotovili, da je 2-propanol najbolj ustrezen obarjalni reagent za sintezo CLEAs iz transglutaminaze. Optimalni volumski procent obarjalnega reagenta je bil 92,4 % (v/v), kar znaša 1215 μl. Za optimalne rezultate smo uporabili raztopino encima s koncentracijo 150 mg/ml s 100 mg/ml EA. Encimske molekule smo zamrežili z 2 % (v/v) GA ob 3 urnem mešanju. Za boljšo stabilnost encima smo dodali 200 μl 0,1 M NaBH3CN. Ugotovili smo, da lahko tako imobilizirano transglutaminazo kot katalizator reakcije uporabimo trikrat z aktivnostjo encima nad 100 % preden se deaktivira.

Keywords

transglutaminaza;zamreženi encimski skupki;glutaraldehidi;mrežni povezovalci;diplomske naloge;

Data

Language:	Slovenian
Year of publishing:	2017
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[Š. Lesičar]
UDC:	543.645.4:544.478.3(043.2)
COBISS:	21033494
Views:	926
Downloads:	98
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Immobilization of transglutaminase as crosslinked enzyme aggregates (CLEAs)
Secondary abstract:	The purpose of this diploma thesis was a successful synthesis of cross-linked enzyme aggregates (CLEAs) from enzyme transglutaminase. The aim was to achieve the maximum possible efficiency of immobilization and the highest remaining activity of transglutaminase. Synthesis of CLEAs consisted of precipitation of the enzyme in chosen precipitant and cross-linking of the enzyme with cross-linking reagent glutaraldehyde (GA). The best precipitant and its optimal volume for synthesis of CLEAs was selected in order to achieve the highest results and we optimized various parameters in process of crosslinking such as concentration of GA and sodium cyanoborohydride (NaBH3CN), concentracion of stabilization proteins bovine serum albumine (BSA) and egg albumin (EA) and concentration of enzyme transglutaminase. The results of diploma thesis shows that the optimal precipitant for synthesis of CLEAs from transglutaminase is 2-propanol with volume percent of 92,4 % (v/v), so the volume of percipitant is 1215 μl. 150 mg/ml of enzyme transglutaminase with 100 mg/ml of EA was used for 3 hours long process of cross-linking with 2% (v/v) GA. We added 200 μl NaBH3CN for stabilization of the enzyme. We also realized that we can reuse the immobilized transglutaminase three times before its activity falls under 100% and the enzyme deactivates.
Secondary keywords:	transglutaminase;immobilization;cross-linked enzyme aggregates;glutaraldehyde;cross-linked agent;
URN:	URN:SI:UM:
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	XII, 40 str.
ID:	10859960