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master's thesis
Purinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija
Zagreb: University of Zagreb, Faculty of Science, 2018. urn:nbn:hr:217:325692
Gucunski, Karolina
University of Zagreb
Faculty of Science
Department of Chemistry

Institutional repository: Repository of the Faculty of Science

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Gucunski, K. (2018). Purinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:325692

Gucunski, Karolina. "Purinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:325692

Gucunski, Karolina. "Purinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija." Master's thesis, University of Zagreb, Faculty of Science, 2018. https://urn.nsk.hr/urn:nbn:hr:217:325692

Gucunski, K. (2018). 'Purinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija', Master's thesis, University of Zagreb, Faculty of Science, accessed 04 November 2024, https://urn.nsk.hr/urn:nbn:hr:217:325692

Gucunski K. Purinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2018 [cited 2024 November 04] Available at: https://urn.nsk.hr/urn:nbn:hr:217:325692

K. Gucunski, "Purinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2018. Available at: https://urn.nsk.hr/urn:nbn:hr:217:325692

Metadata
TitlePurinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija
Title (english)Purine nucleoside phosphorylase from bacterium Helicobacter pylori strain 26695: expresion, purification and characterisation
AuthorKarolina Gucunski
MentorMarija Luić (mentor)
Committee memberJasmina Rokov Plavec (predsjednik povjerenstva)
Committee memberIva Juranović Cindrić (član povjerenstva)
Committee memberBiserka Prugovečki (član povjerenstva)
Committee memberMarija Luić (član povjerenstva)
Committee memberAleksandra Maršavelski (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Chemistry)
Zagreb
Defense date and country2018-09-28, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Chemistry
Abstract
Cilj ovog rada je proĉišćavanje, karakterizacija i kristalizacija enzima purinske nukleozidne fosforilaze (PNPaza) iz bakterije Helicobacter pylori. PNPaza katalizira reverzibilno fosforolitiĉko cijepanje glikozidne veze ribo- i deoksiribonukleozida, u prisutnosti fosfata kao sekundarnog supstrata u svrhu nastajanja purinske baze i riboze(deoksiriboze)-1-fosfata. Neophodan je enzim kod sinteze purina iz metaboliĉkog otpada, jedinog aktivnog puta za sintezu purina u bakteriji H. pylori. Optimizirana je ekspresija PNPaze u bakteriji Escherichia coli. Prekomjerno eksprimirana PNPaza je proĉišćena anionskom izmjenjivaĉkom kromatografijom i afinitetnom kromatografijom na koloni s vezanim inhibitorom. Aktivnost proĉišćenog enzima ispitana je spektrofotometrijski uz razliĉite primarne supstrate. OdreĊeni su kinetiĉki parametri i ispitana je ovisnost aktivnosti PNPaze o pH i temperaturi reakcijske smjese. Postavljeni su eksperimenti kristalizacije s proĉišćenim enzimom metodom difuzije para u visećoj kapi u razliĉitim uvjetima.
Abstract (english)
The goal of this thesis is to purify, characterise and crystallise purine nucleoside phosphorylase (PNPase) from Helicobacter pylori. PNPase catalyses reversible phosphorolytic cleavage of glycoside bond of ribo- and deoxyribonucleosides, in the presence of phosphate as a secondary substrate to produce purine base and ribose(deoxyribose)-1- phosphate. It is required for purine synthesis through salvage pathway, the only active pathway for purine synthesis in H. pylori. The optimal conditions were found for recombinant expression of PNPase in Escherichia coli. Overexpressed PNPase was subsequently purified by anionic exchange chromatography and affinity chromatography on the column with the inhibitor bound. The activity of the purified enzyme was tested spectrophotometrically with different primary substrates. The kinetic parameters were determined as well as the change of PNPase activity with pH and temperature. The hanging drop vapour diffusion method was applied for crystallisation of the purified enzyme.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:325692
Study programmeTitle: Graduate university studies in chemistry; specializations in: Research programme
Course: Research programme
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2018-11-26 10:25:20