Določanje molskih mas ▫$\alpha$▫-amilaze, ▫$\beta$▫-glukozidaze, proteaze in lipaze z metodo gelske kromatografije

magistrsko delo študijskega programa II. stopnje

Jasmina Heric (Author), Maja Leitgeb (Mentor), Maja Čolnik (Co-mentor)

Abstract

Gelska kromatografija je vrsta tekočinske kromatografije in je metoda za ločevanje molekul glede na molekulsko maso. Za gelsko filtracijo uporabljamo steklene kolone, napolnjene s stacionarno (trdno) fazo z enakomerno velikostjo por. Volumen oz. prostor med kroglicami gela (prazen volumen kolone) zapolnjuje mobilna (tekoča) faza. Namen magistrske naloge je bil s pomočjo gelske kromatografije določiti molske mase encimov ▫$\alpha$▫-amilaze, ▫$\beta$▫-glukozidaze, proteaze in lipaze. Najprej smo določili skupno koncentracijo proteinov v vzorcih ter določili elucijsi profil encimov. S pomočjo označevalcev znanih molekulskih mas (metilenskega modrila in dekstran modrega) smo izrisali umeritveno krivuljo za določanje molskih mas in nato določili molske mase encimov ▫$\alpha$▫-amilaze, ▫$\beta$▫-glukozidaze, proteaze in lipaze. Ugotovili smo, da je eksperimentalno določena molska masa ▫$\alpha$▫-amilaze je 54,6 kDa, ▫$\beta$▫-glukozidaze 112,2 kDa, proteaze 78,3 kDa in lipaze 26,6 kDa. Rezultate eksperimentalno določenih molskih mas smo primerjali s podatki o molskih masah najdenih v literaturi. Nato smo vse štiri encime združili v mešanico 4 encimov, katerim smo prav tako določili skupno koncentracijo proteinov s pomočjo Bradfordove metode in nato določili aktivnost posameznih encimov ▫$\alpha$▫-amilaze, ▫$\beta$▫-glukozidaze, proteaze in lipaze v mešanici 4 encimov.

Keywords

magistrska dela;gelska kromatografija;molska masa;▫$\alpha$▫-amilaza;▫$\beta$▫-glukozidaza;lipaza;proteaza;

Data

Language:	Slovenian
Year of publishing:	2019
Typology:	2.09 - Master's Thesis
Organization:	UM FNM - Faculty of Natural Sciences and Mathematics
Publisher:	[J. Heric]
UDC:	543.544.153(043.2)
COBISS:	24848392
Views:	1643
Downloads:	0
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	Determination of the molecular weight of ▫$\alpha$▫-amylase, ▫$\beta$▫-glucosidase, protease and lipase by gel filtration
Secondary abstract:	Gel chromatography is a type of liquid chromatography and a method for separating molecules by molecular weight. For gel filtration, we use glass columns filled with a stationary (solid) phase with a uniform pore size. Volume or the space between the gel balls (empty column volume) fills the mobile (liquid) phase. The purpose of the master's thesis was to determine molar masses of ▫$\alpha$▫-amylase, ▫$\beta$▫-glucosidase, protease and lipase by gel chromatography. We first determined the total concentration of proteins according to the Bradford method by which we determined the elution profile of the enzymes. Using the markers of known molecular weights (methylene blue and blue dextran) we plotted the calibration curve for the determination of molar masses and then determined the molar masses of the ▫$\alpha$▫-amylase, ▫$\beta$▫-glucosidase, protease and lipase enzymes. We found that the molar mass of ▫$\alpha$▫-amylase is 54.6 kDa, ▫$\beta$▫-glucosidase 112.2 kDa, protease 78.3 kDa and lipase 26.6 kDa. The results of experimentally determined molar masses were compared with data on molar masses found in the literature. Then all four enzymes were combined into a mixture of 4 enzymes, which also determined the total protein concentration using the Bradford method, and then determined the activity of the individual enzymes of ▫$\alpha$▫-amylase, ▫$\beta$▫-glucosidase, protease and lipase in a mixture of 4 enzymes.
Secondary keywords:	master theses;gel chromatography;molar mass;▫$\alpha$▫-amylase;▫$\beta$▫-glucosidase;lipase;protease;Univerzitetna in visokošolska dela;
Type (COBISS):	Master's thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za naravoslovje in matematiko, Oddelek za biologijo, katedra za izobraževalno kemijo
Pages:	VII, 55 f.
ID:	11347070