Production of glucose dehydrogenase/cytochrome chimeras by sortagging and characterization of FAD binding constants in GMC-oxidoreductases

master's thesis

• Title (croatian): Proizvodnja glukoza dehidrogenaza/citokrom kimernog enzima pomoću sortaze i karakterizacija konstanti vezanja FAD-a kod GMC-oksidoreduktaza
• Author: Lorna Nikolić
• Mentor: Tonči Rezić (mentor)
• Committee member: Blaženka Kos (član 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: 2021-09-29, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Majority of the growing market for biosensors is driven by medical diagnostics, especially glucose biosensors. Currently, the development of third generation mediatorless biosensors for glucose monitoring is underway. These biosensors employ GMC oxidoreductases as a biological recognition element and an electrochemical transducer for mediatorless detection and quantification of glucose. The problems of direct electron transfer (DET) to electrode, as well as low stability and substrate specificity still have to be overcome and were assesed in this thesis. In order to achieve DET, glucose dehydrogenase (GDH) and cytochrome c were postranslationally conjugated by sortase A to create a chimeric enzyme of improved perfomance mimicking DET capable cellobiose dehydrogenase (CDH). Furthermore, FAD binding constants of native AnGOx, as well as recombinant AnGOX, GcGDH and MtDH, expressed in P. pastoris X33, were determined by deflavination, followed by reflavination of apoenzymes with synthetic FAD. The goal was to create fusion protein capable of DET and to gain insights into the coffactor affinity

Abstract (croatian)

Većinski udio u rastućem tržištu biosenzora zauzimaju potrebe medicinske dijagnostike za detekciju razine glukoze u krvi. Danas se aktivno radi na razvoju glukoznog biosenzora treće generacije, sastavni dio kojeg su GMC oksidoreduktaze, koje provode biokemijsku reakciju i pomoću elektrokemijskog transduktora (pretvarača signala), direktno i bez nusprodukata, prenose elektrone na elektrodu i omogućavaju detekciju i kvantifikaciju glukoze. Problemi koje je potrebno riješiti da bi ova tehnologija bila što pouzdanija su direktni elektronski transfer (DET) s enzima na elektrodu, te niska stabilnost i specifičnost enzima za supstrat. U svrhu postizanja DET-a proizveden je kimerni enzim koji oponaša elektronski transfer celobioza dehidrogenaze (CDH) povezivanjem glukoza dehidrogenaze (GDH) i citokroma c posttranslacijskom konjugacijom posredstvom sortaze A. Također, ispitane su i uspoređene konstante vezanja kofatora FAD-a GMC oksidoreduktaza od interesa (nativni AnGOX, i rekombinantni AnGOX, GcGDH, MtDH, proizvedeni pomoću P. pastoris X33) deflavinacijom i reflavinacijom pomoću sintetskog FAD-a. Cilj je bio proizvodnja sintetskog fuzijskog proteina s mogućnošću DET-a kako bi stekli uvid u afinitet kofaktora.

• Keywords: glucose biosensors; GMC oxidoreductases; chimeric enzymes; dissociation constant; FAD
• Keywords (english): glukozni biosenzori; GMC oksidoreduktaze; kimerni enzimi; konstanta disocijacije; FAD
• Language: english
• URN:NBN: urn:nbn:hr:159:044091
• 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: 2022-05-26 11:16:56