Production and purification of chimeric enzymes and establishment of deflavination method

master's thesis

• Title (croatian): Proizvodnja i pročišćavanje kimernih enzima i uspostava metode deflavinacije
• Author: Karlo Koraj
• Mentor: Božidar Šantek (mentor)
• 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: 2019-12-12, 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: biosensors; chimeric enzymes; DET; deflavination; GMC oxidoreductases
• Keywords (english): biosenzori; kimerni enzimi; DET; deflavinacija; GMC oksidoreduktaze
• Language: english
• URN:NBN: urn:nbn:hr:159:088556
• 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: 2019-12-31 09:38:34