Analiza stabilnosti formijat dehidrogenaze i koenzima NADH u niskotemperaturnim eutektičkim otapalima

master's thesis

• Title (english): Analysis of formate dehydrogenase and NADH coenzyme stability in deep eutectic solvents
• Author: Maja Nikolić
• Mentor: Marina Cvjetko Bubalo (mentor)
• Mentor: Marijan Logarušić (pomoć pri izradi, neposredni voditelj)
• Committee member: Ana Jurinjak Tušek (predsjednik povjerenstva)
• Committee member: Ivana Radojčić Redovniković (član povjerenstva)
• Committee member: Davor Valinger (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Cell Technology, Application and Biotransformations); Zagreb
• Defense date and country: 2024-11-21, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Ovaj diplomski rad istražuje potencijal primjene niskotemperaturnih eutektičkih otapala (engl. Deep Eutectic Solvents, DES) u biokatalitičkim procesima, kroz proučavanje stabilnosti oksidoreduktivnog enzima formijat dehidrogenaze (FDH) i pripadajućeg nikotinamidnog koenzima NADH u različitim DES-ovima. Analizom stabilnosti ovih biomolekula u DES-ovima na bazi betaina i kolin-klorida s glicerolom kao donorom vodika pri različitim udjelima vode odnosno pufera, ustanovljeno je da su enzim FDH te koenzim NADH najstabilniji u DES-ovima na bazi kolin-klorida te da udio vode/pufera ima ključan učinak na stabilizaciju. Nadalje, primjenom Spearmanove koleracijske matrice ispitan je utjecaji ključnih fizičkalno-kemijskih parametra (viskoznost, pH, gustoća) te molekularnih deskriptora (sigma profili generirani primjenom softvera COSMOtherm) DES-ova na stabilnost FDH i NADH te se pokazalo kako svi izmjereni parametri, osim pH, značajno utječu na stabilnost ispitivanih biomolekula, dok sigma profili upućuju da DES-ovi bogati regijama akceptora vodika te nepolarnim regijama uspješnije stabiliziraju FDH i NADH. Također, u sklopu istraživanja izrađeni su i matematički modeli koji opisuju utjecaj fizikalnokemijskih parametara i sigma profila DES-ova na stabilnost ispitivanih biomolekula te su se isti pokazali pouzdanima prema vrijednostima za omjer predviđanja i odstupanja (RPD) i prikladnim za kontrolu kvalitete prema vrijednostima za omjer raspona pogreške (RER). Ovom analizom pruženi su novi uvidi u stabilnost enzima i koenzima u niskotemperaturnim eutektičkim otapalima, otvarajući put za njihovu učinkovitiju primjenu u biokatalitičkim procesima.

Abstract (english)

This thesis explores the potential application of Deep Eutectic Solvents (DES) in biocatalysis by examining the stability of the oxidoreductase enzyme formate dehydrogenase (FDH) and its nicotinamide coenzyme NADH in various DES. An analysis of the stability of these biomolecules in DES based on betaine and choline chloride with glycerol as a hydrogen donor, at different water or buffer contents, revealed that the enzyme FDH and coenzyme NADH were most stable in choline chloridebased DESs, and that the water/buffer content has a critical effect on stabilization. Furthermore, by applying a Spearman correlation matrix, the study examined the impact of key physicochemical parameters (viscosity, pH, density) and molecular descriptors (sigma profiles generated using COSMOtherm software) of DESs on the stability of FDH and NADH. It was shown that all measured parameters, except pH, significantly influence the stability of the tested biomolecules, while sigma profiles indicated that DESs rich in hydrogen acceptor and nonpolar regions more effectively stabilize FDH and NADH. Additionally, mathematical models were developed within the research to describe the influence of physicochemical parameters and sigma profiles of DESs on the stability of the tested biomolecules. These models proved reliable based on values for the ratio of prediction to deviation (RPD) and suitable for quality control according to the ratio of the error range (RER). This analysis provides new insights into the stability of enzymes and coenzymes in low-temperature eutectic solvents, paving the way for their more efficient application in biocatalytic processes.

• Keywords: niskotemperaturna eutektička otapala; biokataliza; formijat-dehidrogenaza; NADH; matematičko modeliranje
• Keywords (english): deep eutectic solvents; biocatalysis; formate dehydrogenase; NADH; mathematical modeling
• Language: croatian
• URN:NBN: urn:nbn:hr:159:308718
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-11-25 09:45:08