Production and purification of fungal enzymes

master's thesis

• Title (croatian): Proizvodnja i pročišćavanje fungalnih enzima
• Author: Matea Zrilić
• Mentor: Tonči Rezić (mentor)
• Committee member: Božidar Šantek (predsjednik povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2020-07-01, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Glyoxal oxidase (GLOX) is an extracellular H2O2-generating metalloenzyme produced by various wood-degrading fungi, one of them being Phanerochaete chrysosporium, a white-rot fungus with the ability to completely degrade lignin. In this thesis, fermentation of P. chrysosporium was conducted on cellulose and sawdust from poplar as carbon sources as well as partial characterization of heterologously produced GLOX was performed. The highest activity of the enzyme was reached at pH 6.0 and the amount of copper present in the enzyme was determined using an assay setup based on complex formation with Zincon, where only 52-66% of the enzymes were found to containe a copper ion in their active site. A broad substrate screening with over 15 compounds was carried out and the highest activity was reached for the extracellular metabolite methylglyoxal. Since the detailed reaction mechanism and mode of activation of GLOX is still unclear, a screening method to test potential activators was developed.

Abstract (croatian)

Glioksal oksidaza (GLOX) izvanstanični je metaloenzim koji stvara vodikov peroksid, a proizvode ga različite gljive sa sposobnošću razgradnje drveta. Jedna od njih je i Phanerochaete chrysosporium, gljiva bijele truleži sa sposobnošću da u potpunosti razgradi lignin. U sklopu izrade ovog rada provedena je fermentacija P. chrysosporium na celulozi i piljevini topole kao izvorima ugljika te je provedena i djelomična karakterizacija heterologno proizvedenog GLOX-a. Najveća aktivnost enzima postignuta je pri pH 6.0, a količina bakra prisutna u aktivnom mjestu enzima određena je uz pomoć Zincon reagensa. Izmjerena količina bakra pokazala je da samo 52-66% enzima sadrži iona bakra u aktivnom mjestu. Provedeno je i testiranje širokog spektra supstrata s preko 15 spojeva, pri čemu je najveća aktivnost postignuta sa izvanstaničnim metabolitom, metilglioksalom. Budući da detaljan mehanizam reakcije i način aktivacije GLOX-a još uvijek nije jasan, razvijena je metoda uz pomoć koje su testirani potencijalni aktivatori.

• Keywords: glyoxal oxidase; Phanerochaete chrysosporium; white-rot fungi; lignin degradation
• Keywords (english): glioksal oksidaza; Phanerochaeta chrysosporium; gljive bijele truleži; razgradnja lignina
• Language: english
• URN:NBN: urn:nbn:hr:159:597355
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-07-01 10:04:07