Proučevanje encimske aktivnosti β-galaktozidaze in priprava srednješolskih kemijskih poskusov

Godec, Ajda (avtor), Sinreih Tisnikar, Maša (mentor)

Encimi kot biokatalizatorji uravnavajo na tisoče reakcij, ki omogočajo pretvorbo energije, sinteze in metabolične razgradnje. Znižana ali povišana encimska aktivnost lahko privede do porušenega ravnovesja na celičnem nivoju, kar se na ravni organizma kaže kot pojav bolezenskega stanja. Eno izmed takšnih bolezenskih stanj, ki je direktno povezano s pomanjkanjem encimske aktivnosti, je hipolaktazija oz. laktozna intoleranca. Pri ljudeh nastopi zaradi znižane encimske aktivnosti β-galaktozidaze (laktaze), encima, ki razgrajuje disaharid laktozo do monosaharidnih komponent. Pacienti za lajšanje simptomov uživajo prehranska dopolnila, katerih aktivna komponenta je encim β-galaktozidaza. Osrednja tema eksperimentalnega dela je bila študija encimske aktivnost β-galaktozidaze iz dveh različnih organizmov E.coli (β-Gal-E) in Asp. oryzae (β-Gal-A). Pri poskusih smo prav tako uporabili β-galaktozidazo (Asp.oryzae) izolirano iz komercialno dostopnega prehranskega dopolnila (β-Gal-P). Zanimal nas je vpliv temperature, pH-vrednosti, koncentracij encima in substrata na aktivnost β-galaktozidaze v prisotnosti substrata o-nitrofenil-β-D-galaktopiranozid. V poskusih, kjer smo kot substrat uporabili X-gal, smo kolorimetrično opazovali vpliv temperature in koncentracije encima na količino nastalega obarvanega produkta. Z eksperimenti smo potrdili, da naraščajoče vrednosti temperature reakcijske mešanice ali koncentracije encima, povečajo količino nastalega produkta oz. encimsko aktivnost. β-Gal-A in β-Gal-P sta dosegla maksimalne vrednosti encimske aktivnosti pri pH 5, encim β-Gal-E pa pri pH-vrednosti 7,4. Na osnovi izvedenih poskusov smo pripravili navodila za izvedbo laboratorijskih vaj, ki omogočajo medpredmetno povezovanje znanj biologije in kemije. Prvi sklop pripravljenih poskusov se bolj osredotoči na razumevanje vplivov na encimsko aktivnosti β-galaktozidaze in uporabo spektrofotometra, drug sklop pa na uporabljeno analizno metodo in zgradbo kolorimetra, ki lahko služi kot prototip za razumevanje delovanja drugih spektroskopskih metod.

• Jezik: Slovenski jezik
• Ključne besede: β-galaktozidaza, encimska aktivnost, spektrometrija, srednješolski poskusi
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-162582
• COBISS.SI-ID: 216117251
• Datum objave v RUL: 25.09.2024

GODEC, Ajda, 2024, Proučevanje encimske aktivnosti β-galaktozidaze in priprava  srednješolskih kemijskih poskusov [na spletu]. Magistrsko delo. [Dostopano 11 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=162582

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Investigation of the enzyme activity of β-galactosidase and preparation of student experiments

Izvleček

As biocatalysts, enzymes control thousands of reactions that enable energy conversion, synthesis and metabolic degradation. Reduced or increased enzyme activity can lead to a disturbed balance at the cellular level, which manifests as a disease state at the organism level. One of these disease states that is directly related to the lack of enzyme activity is hypolactasia or lactose intolerance. In humans, it occurs due to reduced enzyme activity of β-galactosidase (lactase), an enzyme that breaks down the disaccharide lactose into monosaccharide components. To alleviate the symptoms, patients take dietary supplements whose active ingredient is the enzyme β-galactosidase. The central topic of the experimental work was the investigation of the enzymatic activity of β-galactosidase from two different organisms E.coli (β-Gal-E) and Asp.oryzae (β-Gal-A). In the experiments, we also used β-galactosidase (Asp.oryzae) isolated from a commercially available dietary supplement (β-Gal-P). We were interested in the influence of temperature, pH, enzyme and substrate concentrations on β- galactosidase activity in the presence of o-nitrophenil-β-D-galactopiranoside. In experiments using X-gal as substrate, the effect of temperature and enzyme concentration on the amount of colored product formed was observed colorimetrically. Through experiments, we have confirmed that increasing values of the temperature of the reaction mixture or the concentration of the enzyme increase the amount of product formed or enzyme activity. β-Gal-A and β-Gal-P reached maximum values of enzyme activity at pH 5, and β-Gal-E enzyme at pH 7.4. Based on the experiments, we prepared instructions for conducting laboratory exercises that enable cross-curricular integration of biology and chemistry knowledge. The first set of prepared experiments focuses more on understanding the effects on the enzymatic activity of β-galactosidase and the use of the spectrophotometer, while the second set focuses on the analytical method used and the construction of the colorimeter, which can serve as a prototype for understanding the operation of other spectroscopic methods.

• Ključne besede: β-galactosidase, enzyme activity, spectrometry, high school laboratory experiments