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master's thesis
Production and purification of chimeric enzymes and establishment of deflavination method
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. urn:nbn:hr:159:088556
Koraj, Karlo
University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology

Institutional repository: Repository of the Faculty of Food Technology and Biotechnology

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Koraj, K. (2019). Production and purification of chimeric enzymes and establishment of deflavination method (Master's thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:088556

Koraj, Karlo. "Production and purification of chimeric enzymes and establishment of deflavination method." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. https://urn.nsk.hr/urn:nbn:hr:159:088556

Koraj, Karlo. "Production and purification of chimeric enzymes and establishment of deflavination method." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2019. https://urn.nsk.hr/urn:nbn:hr:159:088556

Koraj, K. (2019). 'Production and purification of chimeric enzymes and establishment of deflavination method', Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 22 January 2025, https://urn.nsk.hr/urn:nbn:hr:159:088556

Koraj K. Production and purification of chimeric enzymes and establishment of deflavination method [Master's thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2019 [cited 2025 January 22] Available at: https://urn.nsk.hr/urn:nbn:hr:159:088556

K. Koraj, "Production and purification of chimeric enzymes and establishment of deflavination method", Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2019. Available at: https://urn.nsk.hr/urn:nbn:hr:159:088556

Metadata
TitleProduction and purification of chimeric enzymes and establishment of deflavination method
Title (croatian)Proizvodnja i pročišćavanje kimernih enzima i uspostava metode deflavinacije
AuthorKarlo Koraj
MentorBožidar Šantek (mentor)
GranterUniversity 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 country2019-12-12, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
Glucose biosensors are important analytical devices, which most often use glucose dehydrogenase or glucose oxidase, both from the GMC oxidoreductases family, as a biological recognition element. The use of enzymes in biosensors is hampered in many ways, e.g. by low activity and stability of enzymes, substrate specificity and most importantly by an insufficient connection of the bioelement with an electrode. In order to achieve direct electron transfer between the enzyme and electrode, two glucose dehydrogenases were connected to the heme b-containing cytochrome domain from cellobiose dehydrogenase IIA, from Neurospora crassa, which is naturally capable of DET. Chimeric enzymes were produced in Pichia pastoris and then purified. Moreover, flavoenzymes with loss of FAD cofactor by dissociation immediately lose their activity. In order to determine the FAD dissociation constant of GMC oxidoreductases and further improve their FAD loading, a method for deflavination and reconstitution of flavoenzymes was established. Two different GMC oxidoreductases, used in glucose biosensors, were produced in the Pichia pastoris GlycoSwitch strain and the native and reconstituted enzymes were characterized in comparison.
Abstract (croatian)
Biosenzori za mjerenje glukoze su važni analitički uređaji koji najčešće koriste glukoza dehidrogenazu ili glukoza oksidazu, iz obitelji GMC oksidoreduktaza, kao biološki element. Upotreba enzima u biosenzorima ograničena je niskom aktivnosti i stabilnosti enzima, specifičnosti enzima prema supstratu te najvažnije nezadovoljavajućom povezanosti bioelementa s elektrodom. Kako bi se postigao direktni prijenos elektrona s enzima na elektrodu dvije glukoza dehidrogenaze su povezane sa citokrom domenom, koja sadrži hem b. Korištena citokrom domena je iz enzima celobioza dehidrogenaza IIA, iz plijesni Neurospora crassa, koja je prirodno sposobna za direktni transfer elektrona. Kimerni enzimi su proizvedeni u kvascu Pichia pastoris te zatim pročišćeni. Nadalje, flavoenzimi disocijacijom FAD kofaktora automatski gube svoju aktivnost. Uspostavljena je metoda za deflavinaciju i rekonstituciju flavoenzima s ciljem određivanja konstante disocijacije FAD-a za odabrane GMC oksidoreduktaze te poboljšanje enzimskog vezanja FAD-a. Dvije različite GMC oksidoreduktaze, koje se koriste u biosenzorima, su proizvedene u soju kvasca Pichia pastoris GlycoSwitch te su nativni i rekonstituirani enzimi karakterizirani.
Keywords
Keywords (english)
Languageenglish
URN:NBNurn:nbn:hr:159:088556
Study programmeTitle: 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 resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0) icon
Created on2019-12-31 09:38:34