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Interakcije rekombinantnega pernizina z lipidnimi membranami
Gregorič, Klara (Author), Poklar Ulrih, Nataša (Mentor) More about this mentor... This link opens in a new window

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Abstract
Pernizin je zunajcelična proteaza iz hipertermofilne arheje Aeropyrum pernix, aktivna v širokem temperaturnem in pH območju. Zunajcelični encimi so lahko pritrjeni na celično membrano ali pa so prosti. V tej nalogi smo skušali odgovoriti na vprašanje ali je pernizin prost ali pritrjen encim. Rekombinanten pernizin smo proizvedli v bakteriji Escherichia coli, iz arhejskih lipidov pa smo pripravili poenostavljene modele celične membrane, liposome. S sedimentacijskim testom smo pokazali, da se pernizin ne veže na liposome pripravljene iz arhejskih lipidov (arheosomi), te ugotovitve pa smo potrdili z azokazeinskim testom. Z uporabo več metod (fluorescenčne emisijske spektrometrije, cirkularnega dikroizma in merjenje fluorescenčne polarizacije z membransko sondo difenilheksatrienom) smo pokazali, da se sekundarna struktura pernizina ob dodatku liposomov ne spremeni bistveno, kar potrjuje odsotnost hidrofobnih povezav med pernizinom in arheosomi. Iz rezultatov pridobljenih z zgoraj navedenimi metodami smo zaključili, da je pernizin prost encim. Ker so prosti encimi značilni za biofilme, smo preverili ali tudi arheja Aeropyrum pernix tvori biofilme. S cimogramom smo dokazali proteolitično aktivnost v sloju, ki je nastal na površini erlenmajerice med gojenjem arheje, kar dokazuje, da lahko arheje Aeropyrum pernix tvorijo biofilme, katerih sestavni del je pernizin.

Language:Slovenian
Keywords:rekombinanten pernizin, arheja A. pernix, liposomi, sedimentacijski test, azokazeinski test, cirkularni dikroizem
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2019
Publisher:[K. Gregorič]
UDC:577.15:57.088(043.2)
COBISS.SI-ID:9350009 Link is opened in a new window
Views:362
Downloads:159
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Secondary language

Language:English
Title:Interactions of recombinant pernisine with lipid membranes
Abstract:
Pernisine, extracellular protease from hyper thermophilic archaea Aeropyrum pernix, is active in wide temperature and pH range. Secreted (extracellular) enzymes can be attached to the membrane or they can be free. The goal of this thesis was to determine whether pernisine is attached or free enzyme. Recombinant pernisine was produced in Escherichia coli, and liposomes, simplified models of cell membranes were prepared from archaeal lipids. With sedimentation test and azocasein test we showed that pernisine does not bind to the liposomes from archaeal lipids (archeosomes). Using several methods (fluorescence emission spectroscopy, circular dichroism, measurements of fluorescence polarization with membrane probe diphenylhexatriene) we showed that the secondary structure of pernisine in presence of liposomes is not changed significantly, which confirms the absence of hydrophobic interactions between pernisine and archeosomes. Using methods described above we concluded that pernisine is a free enzyme. Free enzymes are significant for biofilms, so we assumed that A. pernix is also forming biofilms. In flasks where archaea were cultivated, we observed a thin film. With zymogram we detected proteolytical activity in this film, which confirms our hypothesis that A. pernix is forming biofilms that consist of pernisine.

Keywords:recombinant pernisine, archaea A. pernix, liposomes, sedimentation test, azocasein test, circular dichroism

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