Vpliv saharoze in sukraloze na stabilizacijo strukture β-laktoglobulina

Pucihar, Samo (author), Lukšič, Miha (mentor)

Eno ključnih področij, ki mu biokemijske vede posvečajo veliko pozornosti, so vodne raztopine proteinov. Tovrstne formulacije bioloških makromolekul vsebujejo poleg komponent pufra tudi številne sotopljence (npr. soli, sladkorje, polimere). Prisotnost sotopljencev lahko vpliva na nativno strukturo proteinov. Proučevanje vplivov sotopljencev na stabilnost proteinov v raztopini in na fazno stabilnost tovrstnih raztopin je pomembno tako s teoretičnega kot s praktičnega vidika (biotehnologija, farmacevtska in prehrambena industrija …). Diplomsko delo obravnava vpliv dveh sladkorjev – saharoze (naravno sladilo) in njenega strukturnega analoga sukraloze (umetno sladilo) – na (de)stabilizacijo strukture β-laktoglobulina (βLG) v acetatnem pufru. S pomočjo meritev cirkularnega dikroizma smo spremljali, kako saharoza in sukraloza vplivata na talilno temperaturo proučevanega globularnega proteina. Za analizo eksperimentalnih podatkov smo uporabili t. i. denaturacijski model dveh ravnotežnih stanj (nativnega in denaturiranega). Pri nizkih vrednostih pH, kjer je večina β-laktoglobulina v monomerni obliki, lahko saharoza pri segrevanju razpade na glukozo in fruktozo. Kot optimalno vrednost pH, pri kateri protein ostane v monomernem stanju, saharoza pa ne hidrolizira, smo določili vrednost 4. V okviru diplomskega dela smo ugotovili, da pri pH = 4 saharoza β-laktoglobulin stabilizira (temperatura polovice prehoda se z naraščajočo koncentracijo saharoze zvišuje), medtem ko sukraloza protein destabilizira (T$_{1/2}$ se z naraščajočo koncentracijo sukraloze znižuje). S povečanjem koncentracije saharoze (0,1–0,5 M) se je talilna temperatura βLG povišala z 89,2 ℃ na 94,0 ℃ (0,5 M raztopina sladkorja), medtem ko je sukraloza (0,1–0,4 M) povzročila znižanje talilne temperature 79,4 ℃ (0,4 M raztopina sladkorja). (De)stabilizacijsko vlogo obeh sladkorjev smo pojasnili v luči njihove kemijske narave (sukraloza je bolj polarna od saharoze) in z vodo mediiranih meddelčnih interakcij (za razliko od saharoze sukraloza povzroči preferenčno dehidracijo proteina).

• Language: Slovenian
• Keywords: protein, β-laktoglobulin, saharoza, sukraloza, cirkularni dikroizem
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2025
• PID: 20.500.12556/RUL-173241
• COBISS.SI-ID: 257653251
• Publication date in RUL: 15.09.2025

Secondary language

• Language: English
• Title: The influence of sucrose and sucralose on structural stabilization of β-lactoglobulin

Abstract

Aqueous protein solutions represent a key area of biochemical research. Such formulations of biological macromolecules contain buffer species and various cosolutes (e.g. salts, sugars, polimers …). The presence of cosolutes can influence the native structure of protein. It is therefore crucial to characterize the cosolutes' effects on protein's conformational and phase stability from a theoretical and practical standpoint (biotechnology, pharmaceutical and food industry etc.). The following diploma thesis offers insight into effects of two sugars – sucrose (natural sweetener) and its structural analog sucralose (artificial sweetener) – on (de)stabilization of β-lactoglobulin (βLG) structure in acetate buffer. Circular dichroism was used to measure how sucrose and sucralose influence the globular protein's melting temperature. A so-called Two-state denaturation model was employed for data analysis (the two states being native and denaturated protein state). At lower pH values, where β-lactoglobulin is mainly found in monomeric form, sucrose tends to hydrolyze upon heating into glucose and fructose. Therefore pH = 4 was determined to be an optimal value at which hydrolysis is prevented while the protein remains primarily in its monomeric form. The thesis reports that at pH = 4 sucrose stabilizes (i.e. protein's melting temperature rises with increasing sucrose concentration), while sucralose destabilizes (T$_{1/2}$ decreases with rising sucralose concentration) the protein. With increasing sucrose concentration (0.1–0.5 M), the melting temperature of βLG increased from 89.2 ℃ to 94.0 ℃ (for the 0.5 M sugar solution), while sucralose (0.1–0.4 M) caused a decrease in the melting temperature to 79.4 ℃ (for the 0.4 M sugar solution). Their respective influences were interpreted based on their chemical properties (sucralose exhibits greater polarity than sucrose) and water-mediated interactions (sucralose preferentially dehydrates the protein unlike sucrose).

• Keywords: protein, β-lactoglobulin, sucrose, sucralose, circular dichroism