Optimiranje in kinetične študije encimsko katalizirane hidrolize laktoze

magistrsko delo

Gregor Čokl (Author), Andreja Goršek (Mentor), Darja Pečar (Co-mentor)

Abstract

Namen magistrskega dela je bila raziskava procesa imobilizacije encima galaktozidaze na trdi nosilec, ki je bil v našem primeru silikagel. Študija je bila usmerjena predvsem na presnovo mlečnega sladkorja-laktoze ter vzporedno še na kvantitativno analizo vsebnosti prostih encimov v raztopini, s čimer se je določala stopnja imobilizacije encima. Delo je potekalo v dveh fazah, od katerih je bila prva proces imobilizacije encima in druga reakcija tako pripravljenega nosilca z laktozno puferno raztopino. Pred samim procesom imobilizacije je bilo potrebno pripraviti ustrezni pufer. Izbrali smo fosfatni pufer (K2HPO4/KH2PO4) z različnimi koncentracijami in pH, ki smo mu dodali določeno količino laktoze. Med procesom imobilizacije je bilo najprej pomembno čiščenje silikagela ter šele nato nanos APTES-a, glutaraldehida in encima. Reakcija je potekala v čaši (šaržni proces) pri konstantni temperaturi (40 oC) v dveh zaporednih serijah po 2 uri. Vzorce smo odvzemali na vsakih 15 minut, ki smo jih kuhali, da smo denaturirali encim. Vzorce za določitev prostih proteinov smo jemali le na koncu vsake serije. Po reakciji je sledila analiza vzorcev s spektometrom. Določili smo absorbanco, iz katere smo s pomočjo predhodno narejene umeritvene krivulje, tako za proteinske kot glukozne teste, izračunali ustrezne koncentracije. Iz rezultatov je razvidno, da je maksimalna presnova pri pH = 7,0, kar je v skladu z objavljenimi študijami. Proteinski testi pa so pokazali nižjo vsebnost encima v raztopini pri nižjem pH, kar se prav tako sklada z literaturnimi podatki.

Keywords

hidroliza;silikagel;laktoza;beta-galaktoziaza;

Data

Language:	Slovenian
Year of publishing:	2014
Typology:	2.09 - Master's Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[G. Čokl]
UDC:	66.094.941:661.183.7:637.345(043.2)
COBISS:	18402582
Views:	1353
Downloads:	192
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	OPTIMIZING AND KINETIC STUDIES OF THE ENZYME CATALYZED HYDROLYSIS LACTOSE
Secondary abstract:	The aim of this work was to investigate the immobilization process of enzyme galactosidase on solid silica gel carrier (support). The study was mainly focused on the conversion of lactose and quantitative analysis of the free enzyme content in solution, in order to determine the degree of enzyme immobilization. The work was carried out in two steps, the first was enzyme immobilization process and the second reaction of the prepared carrier with lactose buffer solution. An appropriate buffer had to be prepared before the immobilization. We have chosen phosphate buffer (K2HPO4 / KH2PO4) with different concentrations and pH values. Afterwards the weighed amount of lactose was added. The first major important step during immobilization process was cleaning of silica gel and then application of individual components such as APTES, glutaraldehyde (GA) and the enzyme. The reaction was carried out in beaker (batch process) at a constant temperature (40 °C) in two sequentia 2 hours series. Samples were taken every 15 minutes, followed by cooking for protein denaturation. Protein samples were taken only at the end of each series. After the reaction, analysis with spectrometer was carried out. From measured absorbance appropriate concentrations for protein and glucose were obtained using calibration curves. Results show the maximum conversion at pH = 7,0, which is consistent with published studies. Protein tests have showed a lower content of the enzyme in solution at a lower pH, which is also consistent with the literature data.
Secondary keywords:	hydrolysis;beta-galactosidase;sislicagel;lactose;
URN:	URN:SI:UM:
Type (COBISS):	Master's thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	VIII, 35 str.
ID:	8690649