Kinetika biokatalitičke sinteze N-acetilneuraminske kiseline

master's thesis

• Title (english): Kinetics of biocatalytic synthesis of N-acetylneuraminic acid
• Author: Nikolina Jušić
• Mentor: Zvjezdana Findrik Blažević (mentor)
• Committee member: Zvjezdana Findrik Blažević (predsjednik povjerenstva)
• Committee member: Martina Sudar (član povjerenstva)
• Committee member: Marija Vuković Domanovac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2023-09-13, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

U ovom radu provedena je reakcija kondenzacije N-acetilmanozamina i fosfoenolpiruvata uz metalni kofaktor Co2+. Reakcija je katalizirana s enzimom PNH5, umjetno dobivenim na temelju sličnosti s sintazom neuraminske kiseline iz bakterije Neisseria meningitidis.
Kao produkt nastaje N-acetilneuraminska kiselina. Provedena su ispitivanja kako bi se okarakterizirao enzim PNH5 u navedenoj reakciji. Ispitivan je utjecaj pH i koncentracije metalnog kofaktora, kao i utjecaj koncentracije supstrata, odnosno produkta na aktivnost enzima. Ispitana je i mogućnost povratne reakcije, odnosno pretvorbe novonastalog produkta natrag u supstrat. Također, proučavana je i stabilnost PNH5 enzima pri uvjetima skladištenja tijekom 7 tjedana. U programskom paketu SCIENTIST napravljen je model te su izvedene simulacije i procijenjeni kinetički parametri za reakciju. Iz rezultata se može zaključiti da enzimu PNH5 najbolje odgovaraju uvjeti rada pri pH vrijednosti 5.5 te uz Co2+ kao metalni kofaktor. Također, može se uočiti da se fosfoenolpiruvat ponaša kao nekompetitivni inhibitor, dok N-acetilmanozamin i produkt N-acetilneuraminska kiselina ne inhibiraju enzim. Reakcija se odvija isključivo u smjeru nastajanja N-acetilneuraminske kiseline. Iz ispitivanja stabilnosti pri uvjetima skladištenja uočava se da tijekom vremena enzim PNH5 gubi aktivnost i koncentracija proteina pada zato što dolazi do aglomeracije.

Abstract (english)

The condensation between N-acetylmannosamine and phosphoenolpyruvate performed with the metal cofactor Co2+, was carried out in this work. The reaction was catalyzed by PNH5 enzyme, artificially obtained based on its similarity to the N-acetylneuraminic synthase enzyme from the Neisseria meningitidis bacteria. As a product, N-acetylneuraminic acid was formed. Experiments were carried out to characterize the PNH5 enzyme in the investigated reaction. Experiments on the influence of pH and metal cofactor concentration, as well as the influence of substrate and product concentration on the PNH5 enzyme activity, were carried out. The possibility of a reverse reaction, i.e. the conversion of the newly formed product back into the substrate, was also examined. Also, the stability of the PNH5 enzyme under storage conditions for 7 weeks was studied. A kinetic model was created and the kinetic parameters were estimated by using SCIENTIST software package. It can be concluded from the results that the PNH5 enzyme performed best when at a pH value of 5.5 and with Co2+ as a metal cofactor. From the kinetics of the reaction, non-competitive inhibition of the enzyme with phosphoenolpyruvate was observed, while N-acetylmannosamine and the product N-acetylneuraminic acid do not inhibit the enzyme. Also, it can be concluded that there was no reverse reaction because there was no conversion of the newly formed product into a substrate. From the stability in storage conditions experiments, it was observed that over time the PNH5 enzyme loses its activity and protein concentration drops because aggregation occurs.

• Keywords: kinetika; biokataliza; N-acetilneuraminska kiselina; enzim PNH5; model
• Keywords (english): kinetics; biocatalysis; N-acetylnauraminic acid; PNH5 enzyme; model
• Language: croatian
• URN:NBN: urn:nbn:hr:149:066685
• Project: Number: 956631; Title: C-C Bond Formation Using Top Performing Enzymes; Acronym: CC-Top; Leader: Zvjezdana Findrik Blažević; Jurisdiction: eu; Funder: EK; Funding stream: H2020
• Study programme: Title: Applied Chemistry - Graduate study; specializations in: Environmental chemistry and green technologies, Advanced materials and technologies, Applied organic chemistry; Course: Applied organic chemistry; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra primijenjene kemije (magistar/magistra primijenjene kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-09-01
• Created on: 2023-09-21 11:30:47