Imobilizacija encima alkohol dehidrogenaze na dekstranske magnetne nanodelce

diplomsko delo univerzitetnega študijskega programa I. stopnje

Ela Pintar (Author), Maja Leitgeb (Mentor), Katja Heržič (Co-mentor)

Abstract

Imobilizacija encima na nosilec vpliva na stabilnost in aktivnost encima. V diplomskem delu je prikazana študija imobilizacije encima alkohol dehidrogenaze (ADH) na magnetne maghemitne nanodelce, prevlečene s karboksimetil dekstranom (CM-dekstranom). Imobiliziranemu encimu smo aktivnost določali po encimskem testu z merjenem absorbance s spektrofotometrom pri 340 nm, preostalo specifično aktivnost pa izračunali s primerjanjem aktivnosti imobiliziranega z aktivnostjo prostega encima. Določevali smo tudi učinkovitost imobilizacije, in sicer z merjenjem koncentracije proteinov po Bradfordovi metodi pri valovni dolžini 595 nm. Cilj je bil ugotoviti optimalne imobilizacijske pogoje za čim višjo preostalo specifično aktivnost imobiliziranega encima. Pogoje smo spreminjali postopoma in v vsakem naslednjem koraku uporabili optimalne pogoje prejšnjega koraka. Osnovna imobilizacija je potekala 2 uri na 20 °C, hitrost stresanja je bila 300 rpm, pH imobilizacijskega medija pa 7.5, na delce smo dodali 4 % (v/v) epiklorohidrina (ECH) in 0.02 mg/mL encima ADH. Na začetku smo raziskovali vpliv koncentracije ECH, ugotovili smo, da dobimo najvišje preostale aktivnosti imobiliziranega encima pri 4 % (v/v). Naslednji parameter je bila temperatura, ki smo jo za optimalne rezultate znižali na 4 °C. Povečali smo tudi hitrost stresanja na 500 rpm. Najvišje preostale aktivnosti imobiliziranega encima za optimalen pH medija, čas imobilizacije in koncentracijo encima pa so ostale enake, kot pri osnovni imobilizaciji, torej pH 7.5, čas imobilizacije 2 uri in koncentracija encima 0.02 mg/mL. Raziskovali smo tudi stabilnost imobiliziranega encima po 3 h, 5 h in 24 h na 20 °C in 40 °C, za encim, imobiliziran v osnovnih in optimalnih pogojih. Preverjali smo tudi možnosti shranjevanja imobiliziranega encima na 4 °C, za osnovno imobilizacijo 22 dni, za optimalno pa 30 dni. V obeh primerih smo ugotovili, da imobiliziran encim dobro ohranja aktivnost tudi po daljšem časovnem obdobju.

Keywords

alkohol dehidrogenaza;CM-dekstran;imobilizacija;maghemitni nanodelci;preostala specifična aktivnost;stabilizacija encima;

Data

Language:	Slovenian
Year of publishing:	2019
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[E. Pintar]
UDC:	577.152.1(043.2)
COBISS:	22263318
Views:	783
Downloads:	80
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Immobilization of alcohol dehydrogenase enzyme on dextran magnetic nanoparticles
Secondary abstract:	Immobilization of an enzyme on a carrier affects the enzyme's stability and activity. This thesis depicts the research on immobilization of alcohol dehydrogenase enzyme (ADH) on maghemite nanoparticles covered in carboxymethyl-dextran (CM-dextran). The activity of the immobilised enzyme was determined with an enzyme test by measuring the absorbance with a spectrophotometer at 340 nm. Remaining specific activity was calculated based on comparison with the free enzyme's activity. Immobilization efficiency was determined by measuring protein concentration with Bradford method at the Bradford monitoring wavelength (595 nm). The aim of the research was to determine optimal immobilization conditions for the highest remaining specific activity of the immobilized enzyme. The conditions were gradually modified and each step used the findings from the previous step. Basic immobilization was carried out for 2 hours at 20°C and 300 rpm shaking speed. Immoblization medium's pH was 7.5, and 4% (v/v) epichlorohydrin along with 0.02 mg/mL ADH enzyme was added to the particles. Initially, the effect of ECH concentration was researched and it was established that the highest values of immobilized enzyme's remaining activity were at 4% (v/v). The next parameter was temperature which was reduced on 4 °C for optimal results. Shaking speed was increased to 500 rpm. The highest values of remaining activities of the immobilized enzyme for optimal medium pH, immobilization time and enzyme concentration remained the same as in basic immobilization: pH 7.5, immobilization time 2 hours and enzyme concentration 0.02 mg/mL. The stability of the immobilized enzyme was established after 3, 5 an 24 hours at 20°C in 40°C for the enzyme immobilized in basic and optimal conditions respectively. The possibility of storing immobilized enzyme at 4°C was investigated for the period of 22 days after basic immobilization and 30 days after optimal immobilization. In both cases it was established that the immobilized enzyme retains its properties well even after a longer time period.
Secondary keywords:	alcohol dehydrogenase;CM-dextran;immobilization;maghemite nanoparticles;remainig specific activity;enzyme stabilization;
URN:	URN:SI:UM:
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	IX, 27 str.
ID:	11050365