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Halohydrin dehalogenases (HHDHs) catalyse enantioselective formation and conversion of epoxides. Their exceptional selectivity, regioselectivity, and ability to employ different nucleophiles make the enzymes attractive for biocatalysis.[1] The enzyme from Agrobacterium radiobacter AD1 (HheC) is usually expressed in E.coli, isolated in TEMG buffer (50 mM Tris-SO4, 10 mM EDTA, 10 % glycerol, and 1 mM mercaptoethanol), and usually is stored as a cell- free extract at -70 °C for at least three months without significant loss of activity.[2] Both storage (before use) and operational stability (during use) are highly relevant for biotechnological applications.[3] Lyophilization is an attractive approach for the longterm storage of enzymes at ambient temperature. It also facilitates the use of enzymes in non-aqueous media.[4] In this work, the storage stability of HheC isolated in TEMG and TEM buffer is evaluated at room temperature. With a focus on establishing the optimal lyophilized formulation, different additives were tested for enzyme stabilization during lyophilization. Optimal conditions for the lyophilization of several HHDHs have been investigated. Lyophilized HheC was incubated at 50 °C to intensify the differences induced by additives. Enzyme activity is determined by following the absorbance at 310 nm (Figure 1).

Halogenhidrin-dehalogenaze (HHDH) su enzimi koji kataliziraju nukleofilno otvaranje epoksida koristeći niz malih aniona kao što su azid, cijanid, cijanat i dr. Upotrebom ovih enzima moguće je pripraviti enantiomerno čiste fluorirane građevne blokove čija je dostupnost od velikog značaja pri razvoju lijekova u farmaceutskoj industriji. U okviru ovog rada, provedena je prekomjerna ekspresija 7 različitih HHDH, divljih tipova (HheA, HheB i HheC) i genetski modificiranih varijanti (HheA-N178A, HheC-T134V, HheC-W249P i HheC-W4). Uspješnost prekomjerne ekspresije proteina potvrđena je SDS-poliakrilamidnom elektroforezom. Ispitana je katalitička aktivnost genetski modificiranog enzima HheA-N178A u reakcijama azidolize 5 fluoriranih derivata stiren oksida različitog položaja supstitucije (1a-1e). HheA-N178A je pokazala nisku do vrlo visoku S-enantioselektivnost prema ispitanim supstratima (E = 2 – 165). Za visoku enantioselektivnost reakcije presudan je supstituent na para-položaju, a izrazito nepovoljan utjecaj na enantioselektivnost ima metilna skupina kao drugi supstituent na kiralnom centru supstrata. Halohydrin dehalogenases (HHDH) are enzymes that catalyze the nucleophilic opening of epoxides using a number of anionic nucleophiles, including azide, cyanide, nitrite, cyanate, etc. The use of HHDH gives possibility to prepare enantiomerically pure fluorinated building blocks whose availability is of great importance in pharmaceutical industry. In this thesis, overexpression of 7 different HHDH, wild-type (HheA, HheB and HheC) and genetically modified variants (HheA-N178A, HheC-T134V, HheC-W249P and HheC-W4) was performed. The success of protein overexpression was determined by SDS-polyacrylamide electrophoresis. Catalytic activity of genetically modified halohydrin dehalogenase HheA-N178A in reactions of azidolysis of 5 fluorinated styrene oxide derivatives of different substitution positions (1a-1e) was explored. HheA-N178A showed low to very high S-enantioselectivity towards the tested substrates (E = 2 - 165). Para-position of substituent is crucial for the high enantioselectivity of the reaction, and the methyl group has an extremely unfavorable effect on the enantioselectivity as a second substituent at the chiral center of substrate.

Questa tesi analizza la figura femminile nelle opere di Pier Paolo Pasolini. L’analisi è basata su tre seguenti tragedie: Calderón, Porcile e Orgia. Nella tesi è indicata la breve analisi delle tre tragedie: Calderón, Porcile e Orgia. Lo scopo di questa tesi era di presentare in quale maniere l’autore presenti i personaggi femminili, e quali tipi delle figure femminili ci siano. Prima di tutto era importante mostrare il contesto nel quale appaiono i personaggi femminili e in quale maniera la percezione sociale di una donna, cioè di quello come dovrebbe essere, si rifletta nella rappresentazione letteraria dei personaggi femminili. Per aggiungere il contesto più ampio nella tesi si presenta la vita personale dello scrittore e il suo opus letterario che ci aiuta molto nella comprensione e nell’analisi delle tragedie scelte. Ovaj rad analizira figuru ženskog lika u izabranim tragedijama Pier Paola Pasolinija. Analiza se temelji na tri izabrane tragedije, a to su: Calderón, Porcile i Orgia. U radu se prvo iznosi kratka analiza tragedija. Nadalje je cilj rada bio prikazati na koji način autor portretira ženske likove, te koje sve tipove ženskih likova pronalazimo u njegovim dramama. Prije svega važno je bilo prikazati kontekst unutar kojeg se pojavljuju ženski likovi i na koji se način društveno poimanje ženskog tj. onoga što ona treba biti, reflektira u literarnom prikazu ženskih likova. Zbog dodavanja šireg konteksta izabranih tragedija u radu se dotičemo autorova osobnog života i književnog opusa. This thesis discusses the female figure in the selected tragedies written by Pier Paolo Pasolini. The analysis of the theme of this thesis is based on the three selected tragedies: Calderón, Porcile and Orgia. The main aim of the thesis is to present the way in which the author portrays the female characters and which types of female characters do we find. Furthermore, it was important to show the context in which the female characters appear and how the social perception of women, that is what and how they should be, are reflected in the literary representation of female characters. Equally, to give a broader context to the theme, the thesis gives a brief summary of personal life of the author and his literary production, which is of great help for the comprehension of the chosen tragedies.