Izolacija i pročišćavanje enzima hidrogenaze porijeklom iz Ralstonia eutropha H16

master's thesis

• Title (english): Isolation and purification of hydrogenase from Ralstonia eutropha H16
• Author: Sara Anđelović
• Mentor: Anita Šalić (mentor)
• Mentor: Ana Jurinjak Tušek (komentor)
• Committee member: Anita Šalić (predsjednik povjerenstva)
• Committee member: Bruno Zelić (član povjerenstva)
• Committee member: Ana Jurinjak Tušek (član povjerenstva)
• Granter: University of Zagreb; Faculty of Chemical Engineering and Technology; Zagreb
• Defense date and country: 2024-09-25, Croatia
• Scientific / art field, discipline and subdiscipline: TECHNICAL SCIENCES; Chemical Engineering

Abstract

Jedan od ključnih enzima u enzimatskoj proizvodnji biovodika je enzim hidrogenaza.
U organizmima kao što su bakterije, alge i biljke mogu se pronaći različite vrste hidrogenaza, pri čemu su najzastupljenije [FeFe]-hidrogenaze i [NiFe]-hidrogenaze. U stanicama bakterije Ralstonia eutropha H16 prisutne su tri različite [NiFe]-hidrogenaze tolerantne na kisik uključene u transport H2 što ovu bakteriju čini izvrsnim izvorom hidrogenaza.
Cilj ovog istraživanja bio je heterotrofno uzgojiti stanice R. eutropha H16 na četiri različite sintetske podloge. Tijekom uzgoja praćene su promjene koncentracije biomase i supstrata,
a najbolji rast stanica zabilježen je na sintetskoj podlozi koja je sadržavala 4 g/L fruktoze. Nakon uzgoja stanica, provedena je optimizacija ultrazvučnog razbijanja stanica korištenjem Box-Behnken dizajna eksperimenta s tri varijable (amplituda ultrazvuka, koncentracija stanica i vrijeme razbijanja) na tri razine s ciljem oslobađanja što veće koncentracije proteina.
Kada je proces proveden pri optimalnim procesnim uvjetima (amplituda ultrazvuka 40 %, vrijeme razbijanja 25 min i koncentracija stanica 3 mg/mL) dobivena maksimalna koncentracija proteina u uzorku iznosila je 2,483 ± 0,110 mg/mL.

Abstract (english)

One of the key enzymes in the enzymatic production of biohydrogen is the enzyme hydrogenase. Various types of hydrogenases are found in organisms such as bacteria, algae and plants, with [FeFe]-hydrogenases and [NiFe]-hydrogenases being the most abundant. In the cells of the bacterium Ralstonia eutropha H16, there are three different oxygen-tolerant [NiFe]-hydrogenases involved in H2 transport, which makes this bacterium an excellent source of hydrogenases. The aim of this research was to heterotrophically grow R. eutropha H16 cells on four different synthetic substrates. During cultivation, changes in the concentration of biomass and substrate were monitored, and the best cell growth was recorded on a synthetic medium containing 4 g/L fructose. After cell cultivation, optimization of ultrasonic cell disruption (ultrasound amplitude, cell concentration and disruption time) was performed using a three-level experimental Box-Behnken design with three variables with the aim of releasing the highest possible protein concentration. When the process was carried out under optimal process conditions (ultrasound amplitude 40%, disruption time 25 min and cell concentration 3 mg/mL), the maximum protein concentration obtained in the sample was 2.483 ± 0.110 mg/mL.

• Keywords: enzim hidrogenaza; biovodik; Ralstonia eutropha H16; optimizacija razbijanja stanica
• Keywords (english): hydrogenase enzyme; biohydrogen; Ralstonia eutropha H16; cell disruption optimization
• Language: croatian
• URN:NBN: urn:nbn:hr:149:264854
• Project: Number: IP-2022-10-2175; Title: Integrirani mikrosustav za enzimatsku proizvodnju biovodika; Title: Integrated micro-system for enzymatic biohydrogen production; Acronym: MicroBioH2; Leader: Bruno Zelić; Jurisdiction: Croatia; Funder: Hrvatska zaklada za znanost; Funding stream: Research Projects
• Study programme: Title: Chemical Engineering - Graduate study; Study programme type: university; Study level: graduate; Academic / professional title: sveučilišni/a magistar/magistra inženjer/inženjerka kemijskog inženjerstva (sveučilišni/a magistar/magistra inženjer/inženjerka kemijskog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-11-06 13:39:55