Praćenje stabilnosti enzima putem mjerenja raspodjele veličine proteina

undergraduate thesis

• Title (english): Monitoring of enzyme stability by measuring protein size distribution
• Author: Barbara Arambašić
• Mentor: Zvjezdana Findrik Blažević (mentor)
• Committee member: Zvjezdana Findrik Blažević (predsjednik povjerenstva)
• Committee member: Marija Vuković Domanovac (član povjerenstva)
• Committee member: Miroslav Jerković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2022-09-19, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

N-acetilneuraminska kiselina (Neu5Ac) je sijalična kiselina čiju sintezu katalizira enzim N-acetilneuraminat sintaza. N-acetilmanozamin (MaNAc) kondenzacijom s fosfoenolpiruvatom (PEP) daje N-acetilneuraminsku kiselinu (Neu5Ac). Za potrebe ovog rada enzim je pripremljen i pročišćen. Prekomjerna ekspresija proteina neuB, koji kodira N-acetilneuraminat sintazu, postignuta je ubacivanjem plazmida u genetski modificirane stanice Escherichia coli. Bakterijske stanice razbijene su ultrazvukom i pročišćene centrifugiranjem. Dobiveni enzim pročišćen je u koloni s niklom i nitrilotrioctenom kiselinom i skladišten u odgovarajućem puferu na 4°C. Stabilnost enzima sintaze N-acetilneuraminske kiseline (NeuS) je praćena preko specifične aktivnosti, raspodjele veličina čestica i koncentracije proteina.

Abstract (english)

N-acetylneuraminic acid (Neu5Ac) is a sialic acid whose synthesis is catalyzed by the enzyme N-acetylneuramine synthase. N-acetylmannosamine (MaNAc) by condensation with phosphoenolpyruvate (PEP) gives N-acetylneuraminic acid (Neu5Ac). For the purposes of this work the enzyme was prepared and purified. Overexpression of the protein neuB, which encodes N-acetylneuramine synthase, was achieved by inserting the plasmid into genetically modified Escherichia coli cells. Bacterial cells were disrupted by ultrasound and purified by centrifugation. The resulting enzyme was purified in a column with nickel and nitrilotriooctenoic acid and stored in the appropriate buffer at 4°C. The stability of N-acetylneuraminate synthase (NeuS) enzyme was monitored through specific activity, particle size distribution and protein concentration.

• Keywords: N-acetilneuraminska kiselina; N-acetilmanozamin; fosfoenolpiruvat; N-acetilneuraminat sintaza; neuB
• Keywords (english): N-acetylneuraminic acid; N-acetylmannosamine; phosphoenolpyruvate; N-acetylneuraminate synthase; neuB
• Language: croatian
• URN:NBN: urn:nbn:hr:149:486322
• 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: Chemical Engineering - Undergraduate study; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/ prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka kemijskog inženjerstva (sveučilišni/a prvostupnik/ prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka kemijskog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-09-19
• Created on: 2023-02-17 13:22:46