20.500.12556/DKUM-46788 OPTIMIRANJE IN KINETIČNE ŠTUDIJE ENCIMSKO KATALIZIRANE HIDROLIZE LAKTOZE OPTIMIZING AND KINETIC STUDIES OF THE ENZYME CATALYZED HYDROLYSIS LACTOSE 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. 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. hidroliza silikagel laktoza beta-galaktozidaza hydrolysis beta-galactosidase silicagel lactose true true false [G. Čokl] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2014-11-07 11:18:42 2014-12-03 15:12:31 2022-05-18 03:08:15 2021-05-05 03:47:13 2014 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 66.094.941:661.183.7:637.345(043.2) 18402582 URN:SI:UM:DK:DSX5CXXN MAG_Cokl_Gregor_2014.pdf MAG_Cokl_Gregor_2014.pdf 1 03B91BD15760BE40B73E8AC8F1847640 cf4a8a559a1ca75829ab1b6f1df76ffd44a949cba97ca25269dfa9d6d7aebc2e 73764926-7c0b-11eb-bb7a-00155d0001ca https://dk.um.si/Dokument.php?lang=slv&id=69077 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0