20.500.12556/RUL-184496 Proteinski inženiring oligomerne različice celulaze bglC z uporabo termostabilne tetramerizacijske domene p53 Protein engineering of oligomeric cellulase bglC using a thermostable p53 tetramerization domain Celulaze klasično delimo v tri skupine encimov, ki sinergistično sodelujejo pri hidrolizi celuloze: endoglukanaze (EC 3.2.1.4), eksoglukanaze (EC 3.2.1.91) in β-glukozidaze (EC 3.2.1.21). Celulaze vseh skupin so ključne za pretvorbe lignoceluloznih materialov in so vsestransko uporabne v industriji. Kompleksna sestava celuloze in omejitve obstoječih encimov pri razgradnji celuloze vseeno predstavljajo izzive pri širši praktični uporabi. Učinkovitejšo uporabo celulaz v industrijskih procesih je mogoče doseči z izboljšanjem katalitične aktivnosti encimov, povečanjem njihove stabilnosti in razumevanjem zahtev za uporabo v specifičnih procesih ter inženirstvom encimov, prilagojenih zanje. Eno izmed področij izboljšav je oligomerizacija. Oligomerni encimi so pogosto odpornejši na ekstremne pogoje, hkrati pa lahko oligomerizacija izboljša adsorpcijo encimov na netopne substrate. V sklopu magistrskega dela smo skušali z uporabo termostabilne tetramerizacijske domene p53 (p53TD_Q331R) kot C-končne fuzije pripraviti tetramerno različico celulaze bglC, endoglukanaze iz bakterije Bacillus subtilis. Zanimalo nas je ali fuzijski protein oligomerizira in ali spremenjeno oligomerno stanje vpliva na aktivnost razgradnje polimernega substrata karboksimetilceluloze. Zapis za fuzijski protein smo pripravili s sestavljanjem po Gibsonu, nato pa izrazili in izolirali obe različici endoglukanaze ter določili njuno oligomerno stanje z uporabo velikostne izključitvene kromatografije in masne fotometrije. Aktivnost obeh različic smo preučevali z uporabo testa DNS ter tako določili pripadajoče kinetične parametre. Tetramerne različice celotnega fuzijskega proteina nam ni uspelo pripraviti, najverjetneje zaradi proteolitičnih cepitev fuzijskega proteina na področju povezovalne regije med domenama celulaze bglC. Proteolitične cepitve in odsotnost tetramerizacije bi lahko vplivale tudi na nižjo navidezno aktivnost fuzijskega proteina v primerjavi z rekombinantno celulazo bglC divjega tipa. Cellulases are classically divided into three groups of enzymes, synergistically involved in cellulose hydrolysis: endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91) and β- glucosidases (EC 3.2.1.21). Cellulases belonging to all three groups are crucial for the conversion of lignocellulosic materials and are useful in various industries. However, complex cellulose structure and limitations of existing enzymes in cellulose degradation restrict broader cellulase use in industrial applications. More efficient use of cellulases for this purpose can be achieved with the improvement of their catalytic activity and stability, as well as by understanding process-specific enzyme requirements and engineering tailored enzymes for these processes. One way of improving cellulase applicability is through oligomerization. Oligomeric enzymes often exhibit greater resistance to extreme conditions; additionally, oligomerization can increase enzyme adsorption to insoluble substrates. As part of this master’s thesis, we aimed to prepare a tetrameric version of cellulase bglC, an endoglucanase from the bacterium Bacillus subtilis, by using a thermostable p53 tetramerization domain (p53TD_Q331R) as a C-terminal fusion. We investigated whether the fusion protein oligomerises and whether the change in oligomeric state influences its activity against the polymeric substrate carboxymethylcellulose. We prepared the fusion protein construct by Gibson assembly and purified both variants of the endoglucanase. We determined the oligomeric state with size exclusion chromatography and mass photometry. Enzymatic activity of both variants was subsequently analysed using the DNS assay, enabling the determination of the corresponding kinetic parameters. We were unable to obtain the intended tetrameric form of the full-length fusion protein, likely due to proteolytic cleavage of the fusion protein in the interdomain region of cellulase bglC. Proteolytic cleavage and the absence of fusion protein tetramerization could explain why the fusion protein exhibited lower apparent activity compared to the recombinant wild-type variant of cellulase bglC. celulaza bglC endoglukanaza celuloza oligomerizacija tetramerizacijska domena p53 cellulase bglC endoglucanase cellulose oligomerization p53 tetramerization domain true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-07-08 14:10:32 2026-07-08 14:10:50 2026-08-21 11:29:06 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25929 288455939 Starc_Gaja_-_Proteinski_inzeniring_oligomerne_razlicice_celulaze_bglC_z_uporabo_termostabilne_te.pdf Starc_Gaja_-_Proteinski_inzeniring_oligomerne_razlicice_celulaze_bglC_z_uporabo_termostabilne_te.pdf 1 CA04AEBE496E3D148A470902B9971343 b66687ac0cd2557cdf77147f5a0b1c7558ddc781ed57cf45633c149316cead44 f967a493-7ac5-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=237358 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0