Purinska nukleozidna fosforilaza iz soja 26695 bakterije Helicobacter pylori: ekspresija, pročišćavanje i karakterizacija

master's thesis

• Title (english): Purine nucleoside phosphorylase from bacterium Helicobacter pylori strain 26695: expresion, purification and characterisation
• Author: Karolina Gucunski
• Mentor: Marija Luić (mentor)
• Committee member: Jasmina Rokov Plavec (predsjednik povjerenstva)
• Committee member: Iva Juranović Cindrić (član povjerenstva)
• Committee member: Biserka Prugovečki (član povjerenstva)
• Committee member: Marija Luić (član povjerenstva)
• Committee member: Aleksandra Maršavelski (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2018-09-28, 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: afinitetna kromatografija s vezanim inhibitorom; anionska izmjenjivaĉka kromatografija; Helicobacter pylori; kinetiĉki parametri; kristalizacijske probe; purinska nukleozidna fosforilaza
• Keywords (english): afinity chromatography with bound inhibitor; anionic exchange chromatography; Helicobacter pylori; kinetic parameters; crystallization probes; purine nucleoside phosphorylase
• Language: croatian
• URN:NBN: urn:nbn:hr:217:325692
• Study programme: Title: 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 resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-11-26 10:25:20