[doktorsko delo]
Miha Bahun (Author), Nataša Poklar Ulrih (Mentor)

Abstract

Encimi, ki izvirajo iz termofilnih organizmov, so zaradi izjemne stabilnosti zaželeni za uporabo v industriji. Eden izmed industrijsko zanimivih encimov je pernizin iz hipertermofilne arheje Aeropyrum pernix. Pernizin je subtilizinu podobna zunajcelična proteinaza, ki izmed ostalih proteinaz izstopa zaradi zmožnosti razgradnje infektivnih prionskih proteinov. Pernizin bi torej bilo možno uporabiti kot sredstvo za čiščenje površin, okuženih s prioni, uporaben pa bi bil tudi v živilski ter usnjarski industriji. Pernizin je tako kot ostali subtilizini po sintezi sprva v neaktivni proobliki, ki se nato avtokatalitično pretvori v aktivno proteinazo. V sklopu doktorskega dela smo proučili proces aktivacije propernizina, pri čemer smo pripravili različne mutante tega proteina in preverili njihovo zmožnost zvitja ter aktivacije. Ugotovili smo, da aktivacija propernizina obsega več stopenj. Sprva se cepi peptidna vez med proregijo in katalitično domeno, kar vodi do nastanka avtoprocesiranega kompleksa. Ta zaradi inhibitornega delovanja nekovalentno vezane proregije še ni proteolitično aktiven. Aktivacija se zaključi z disociacijo avtoprocesiranega kompleksa ter razgradnjo sproščene proregije s katalitično domeno. Za ta končni korak aktivacije so potrebni kalcijevi ioni, ki omogočijo zvitje pernizina v urejeno konformacijo in ga s tem zaščitijo pred avtolizo. Izkazalo se je, da ima ključno vlogo pri stabilizaciji pernizina med aktivacijo edinstvena insercija, ki tvori dodatno mesto za vezavo kalcijevega iona na katalitični domeni. Poleg kalcija so za aktivacijo propernizina potrebne tudi temperature nad 80 °C. Namreč, sproščeno proregijo destabilizirajo zgolj visoke temperature, kar vodi v njeno razgradnjo in zaključek aktivacije. Ugotovili smo, da je glavna vloga proregije pernizina pri aktivaciji inhibicija katalitične domene, saj proregija ni vplivala tudi na zvitje te domene, kot je to sicer primer pri bakterijskih subtilizinih. Optimizirali smo tudi način pridobivanja rekombinantnega pernizina v bakterijah Escherichia coli. Agregacijo propernizina v citoplazmi E. coli smo preprečili z usmeritvijo sintetiziranega propernizina v periplazemski prostor celic, s čimer smo hkrati tudi poenostavili postopek čiščenja te proteinaze. Pernizin, izoliran iz periplazme, je ohranil visoko temperaturno stabilnost ter aktivnost. Pripravili smo tudi več različic pernizina, skrajšanih z N-konca oziroma C-konca, ter preverili njihovo aktivnost. Rezultati kažejo, da so za visoko temperaturno stabilnost pernizina pomembna tudi zunanja področja katalitične domene, v katerih so vezavna mesta za kalcijeve ione.

Keywords

biokemija;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL BF - Biotechnical Faculty
Publisher: M. Bahun
UDC: 577.1/.3:582.233
COBISS: 37466627 Link will open in a new window
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Downloads: 216
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Other data

Secondary language: English
Secondary title: Activation and temperature stability determinants of the serine proteinase pernisin from hyperthermophilic archaeon Aeropyrum pernix K1
Secondary abstract: Enzymes from thermophilic organisms are desired in industrial applications, due to their exceptional stability. One of the industrially relevant enzymes is pernisin from the hyperthermophilic archaeon Aeropyrum pernix. Pernisin is a subtilisin-like proteinase that stands out among other proteinases due to its prion protein degrading activity. Therefore, pernisin might be used for cleaning different surfaces contaminated with prions, as well as in food and leather industry. As for other subtilisins, pernisin is synthesized in its inactive proform, which is then autocatalitically converted into the active proteinase. In this thesis we investigated the activation process of propernisin. We prepared different propernisin mutants and analyzed their folding and activation capability. We found out that propernisin activation proceeds in several steps. First, the peptide bond between the propeptide and the catalytic domain is autocatalitically cleaved, resulting in proteolytically inactive non covalent complex of the propeptide and the catalytic domain. The activation is completed upon dissociation of the autoprocessed complex and degradation of the released propeptide with the catalytic domain. For this final activation step, calcium ions are needed since they enable pernisin to fold into ordered conformation, which is protected against autodegradation. Importantly, the unique insertion that forms an additional calcium ion binding site within the catalytic domain is essential for pernisin stability during the activation. Besides calcium ions, also temperatures above 80 °C are required for the complete activation, as the released propeptide is destabilized at high temperatures only, which eventually leads to propeptide degradation. The major role of the pernisin propeptide is inhibition of the catalytic domain during the activation. Interestingly, the propeptide did not show any chaperoning activity, which is commonly observed in bacterial subtilisins. We also optimized preparation of recombinant pernisin in Escherichia coli. Aggregation of propernisin in cytoplasm was prevented by translocation of produced propernisin in periplasmic space. Consequently, this strategy simplified isolation procedure of this proteinase. Pernisin that was isolated from the periplasm was highly thermostable and active. Additionally, different N-terminally and C-terminally truncated variants of pernisin were prepared and analyzed for their proteolytic activities. Based on our results, the outer regions of the catalytic domain with calcium binding sites are important for pernisin thermostability.
Secondary keywords: Recombinant proteins;Chemical synthesis;Isolation and purification;Enzyme activation;Analysis;Enzyme stability;Subtilisins;History;Archaea;Serine proteases;Methods;Electrophoresis, polyacrylamide gel;Rekombinantni proteini;Kemična sinteza;Izolacija in čiščenje;Aktivacija encima;Analiza;Encimska stabilnost;Subtilizini;Zgodovina;Arheje;Serinske proteaze;Metode;Poliakrilamidna gelska elektroforeza;
Type (COBISS): Doctoral dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Medicinska fak.
Pages: X, 102 str.
ID: 12101439