20.500.12556/RUL-167299 Biosinteza termostabilne serinske proteinaze iz arhej v bakteriji Bacillus subtilis Biosynthesis of archaeal thermostable serine proteinase in Bacillus subtilis Pernizin je subtilizinu podobna serinska proteinaza iz hipertermofilne arheje Aeropyrum pernix K1. Zaradi izjemne temperaturne stabilnosti in zmožnosti razgradnje infektivnih prionskih proteinov, ima pernizin visok potencial za industrijske aplikacije. Praktično uporabo pernizina omejuje nizek donos te proteinaze v izvornem organizmu, kjer je manjši od 0,1 mg na liter kulture. Bakterije B. subtilis so primeren gostiteljski organizem za proizvodnjo rekombinantnih proteinov zaradi poznanih močnih promotorjev in zmožnosti učinkovitega izločanja proteinov iz celic, torej bi lahko predstavljale obetaven ekspresijski sistem za proizvodnjo pernizina. Bakterije B. subtilis naravno proizvajajo proteinazo subtilizin, ki je homologna pernizinu, v količinah do 1 g na liter kulture. V magistrski nalogi smo preverili zmožnost biosinteze rekombinantnega pernizina z bakterijami B. subtilis. Pri tem smo primerjali tri različne promotorje (PaprE, Pveg in Pylb) za nadzor izražanja pernizina. Izmed preizkušenih promotorjev se je kot najustreznejši izkazal Pylb, saj smo z uporabo tega promotorja v gojišču zaznali okoli 40 % višjo aktivnost pernizina v primerjavi s promotorjema PaprE ter Pveg. Proizvedeni rekombinantni pernizin smo tudi izolirali iz gojišča ter ovrednotili njegovo aktivnost in stabilnost. S CD-spektrometrijo in fluorescenčno emisijsko spektrometrijo smo potrdili, da izoliran rekombinantni pernizin ohrani podobno visoko temperaturno stabilnost in aktivnost kot rekombinantni pernizin, proizveden v bakterijah Escherichia coli. Pernisine is a subtilisin-like serine proteinase from the hyperthermophilic archaeon Aeropyrum pernix K1. Due to its exceptional temperature stability and ability to degrade infectious prion proteins, pernisine has a high potential for industrial applications. The practical use of pernisine is limited by the low yield of this proteinase in the parent organism, where it is less than 0.1 mg per liter of culture. One of the suitable host organisms for the production of recombinant proteins are Bacillus subtilis bacteria due to their known strong promoters and efficient protein secretion systems, which means that B. subtlis may represent a promising expression system for the production of pernisine. B. subtilis bacteria naturally produce the proteinase subtilisin, which is homologous to pernisine, at levels up to 1 g per litre of culture. In the master's thesis, we examined production of recombinant pernisine in B. subtilis. We compared three different promoters (PaprE, Pveg and Pylb) for controlling pernisine expression. Among the promoters tested, Pylb appeared the most suitable, as around 40 % higher pernisine activity was detected in medium compared to the other two promoters. We have also isolated the produced pernisine from culture medium and evaluated its activity and stability. Using CD spectrometry and fluorescence emission spectrometry we confirmed that recombinant pernisine isolated from B. subtilis has similar high temperature stability and activity as recombinant pernisine produced with Eschericha coli bacteria. Bacillus subtilis pernizin promotor proteinaza proteolitična aktivnost rekombinantni protein rekombinantno izražanje termostabilnost Bacillus subtilis pernisine promoter proteinase proteolytic activity recombinant expression recombinant protein thermostability true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2025-02-14 07:15:33 2025-02-14 07:15:42 2025-02-15 04:13:18 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 256052 226126851 45713.pdf 45713.pdf 1 A25C50D4EE9C13E29A555DE566B36D12 080f22af5789493465db6cb72693be944a4b5a9b3e5b6a5a158ae34b7ede9c39 3bce458b-ea9a-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=199638 Biotehniška fakulteta 0 0 0