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Proteinski inženjering i razvoj visoko efikasnih metoda za pretraživanje biblioteke gena glukoza-oksidaze iz Aspergillus niger u cilju povećanja enzimske aktivnosti i stabilnosti

Protein engineering and development of high-throughput screening methods for Aspergillus niger glucose-oxidase gene library toward higher enzyme activity and stability.

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2018
Disertacija.pdf (96.35Mb)
IzvestajKomisije18732.pdf (1.740Mb)
Author
Kovačević, Gordana N.
Mentor
Prodanović, Radivoje
Committee members
Gavrović-Jankulović, Marija
Baošić, Rada
Nikodinović-Runić, Jasmina
Metadata
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Abstract
Glukoza-oksidaza (GOx) je vaţan industrijski enzim koji se predominantno koristi kao biokatalizator u industriji hrane za proizvodnju glukonske kiseline, uklanjanje kiseonika i sterilizaciju. U farmaceutskoj industriji i kliničkoj biohemiji se koristi kao biosenzor za određivanje koncentracije glukoze, dok postoje pokušaji da se iskoristi i za proizvodnju biogorivnih ćelija koje proizvode električnu energiju koristeći glukozu i kiseonik iz ljudske krvi u okvirima nanobiotehnologije. Za rasprostranjeniju primenu GOx neophodno je unaprediti neke od njenih osobina kao što su aktivnost, pH optimum, reaktivnost sa kiseonikom i stabilnost. Jedna od metoda kojom se unapređuju proteini je dirigovana evolucija. Ova metoda podrazumeva iterativne tehnike generisanja biblioteka proteinskih mutanata (ili varijanti) i selekciju proteina sa odgovarajućom ţeljenom funkcijom iz ovih biblioteka. U cilju pronalaţenja mutanata GOx iz Aspergillus niger koji su oksidativno stabilniji i aktivniji, u ovom rad...u su razvijene dve metode pretraţivanja bazirane na ekspresiji proteina na površini ćelija kvasca. Pored razvoja metoda pretraţivanja ispitivana je i optimizacija ekspresije rekombinantne GOx u kvascu Pichia pastoris za heterolognu ekspresiju mutanata GOx i njihovu kinetičku karakterizaciju. Oksidacija bočnih ostataka amino kiselina je jedan od glavnih razloga nestabilnosti nativne trodimenzionalne strukture GOx. Posebno osetljiv prema oksidaciji je metionin, koji se prevodi u metionin-sulfoksid čak i pod blagim uslovima u prisustvu kiseonika i kiseoničnih reaktivnih vrsta kao što su vodonik-peroksid, superoksid anjon radikal i drugi. Da bi utvrdili uticaj pozicije metionina na oksidativnu stabilnost GOx, svih 11 metionina je razmatrano za mesto specifičnu mutagenezu...

Glucose-oxidase (GOx) is an important industrial enzyme that is predominantly used as a biocatalyst in the food industry for the production of gluconic acid, oxygen removal and sterilization. In the pharmaceutical industry and clinical biochemistry it is used as a biosensor for determining glucose concentration, while there are attempts to utilize it in the production of biofuel cells that produce electricity using glucose and oxygen from human blood within the framework of nanobiotechnology. For the widespread use of GOx it is necessary to improve some of its properties such as activity, pH optimum, reactivity with oxygen and stability. One of the methods for improving proteins is directed evolution. This method involves iterative techniques for generating libraries of protein mutants (or variants) and selecting proteins with the desired function from these libraries. In order to find GOx mutants from Aspergillus niger that are oxidatively more stable and active, in this work two meth...ods of screening based on protein expression on the surface of yeast cells have been developed. In addition to the development of the screening methods, the optimization of the expression of recombinant GOx in yeast Pichia pastoris for heterologous expression of GOx mutants and their kinetic characterization was studied. Aminoacid side chain oxidation is one of the main causes for structural instability of native tridimensional structure of GOx. Particularly sensitive to oxidation is methionine residue, which is converted to methionine sulfoxide even under mild conditions in the presence of oxygen and oxygen reactive species such as hydrogen peroxide, superoxide anion radical and others. In order to determine the effect of the position of the methionine amino acid residue on the oxidative stability of GOx, all 11 methionines were considered for site directed mutagenesis...

Faculty:
Универзитет у Београду, Хемијски факултет
Date:
26-09-2018
Projects:
  • Allergens, antibodies, enzymes and small physiologically important molecules: design, structure, function and relevance (RS-172049)
Keywords:
glukoza-oksidaza / glucose oxidase / visoko efikasne metode pretraţivanja / oksidativna stabilnost / ekspresija na površini ćelija kvasca / zeleni fluorescentni protein / Pichia pastoris / high-throughput screening / oxidative stability / yeast surface display / green fluorescent protein / Pichia pastoris
[ Google Scholar ]
Handle
https://hdl.handle.net/21.15107/rcub_nardus_10586
URI
http://eteze.bg.ac.rs/application/showtheses?thesesId=6425
https://nardus.mpn.gov.rs/handle/123456789/10586
https://fedorabg.bg.ac.rs/fedora/get/o:19139/bdef:Content/download
http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=50764559

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