Search

Stability of enzymes has a significant impact on their use in an alternative reaction media. Presence of organic solvents or exposure to high temperatures leads to denaturation of enzymes since they have evolved to work in the cellular environment. Lyophilization is as an attractive solution for long-term storage of enzymes at ambient temperature. Also, it can be used as a method of enzyme stabilization for their application in non-aqueous media. Halohydrin dehalogeneses are enzymes that catalyse the formation and conversion of epoxides. Reactions are catalyzed in regioselective and enantioselective manner, making the enzymes attractive for biocatalysis. Enzyme from Agrobacterium radiobacter AD1 (HheC) is usually expressed in E.coli, isolated in TEMG buffer (50 mM Tris-SO4, 2 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. In this work, the stability of HheC is first evaluated in different buffers (TEMG, TEM and Tris-SO4) at different temperatures. With the aim of obtaining the optimal lyophilized formulation, different additives were tested for HheC stabilization. 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.

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.

Halogenhidrin-dehalogenaze (HHDH) su enzimi koji kataliziraju nukleofilno otvaranje epoksida koristeći niz malih aniona kao što su azid, cijanid, cijanat i dr. 1. Reakcije se odvijaju u blagim uvjetima, pri sobnoj temperaturi, neutralnom pH i atmosferskom tlaku, a usto pokazuju visoku regio-i enantioselektivnost te kao takve predstavljaju dobru alternativu klasičnim kemijskim metodama sinteze. Upotrebom ovih enzima moguće je pripraviti niz kiralnihgrađevnihblokova čija je dostupnost od velikog značaja u kemijskoj, farmaceutskoj i agrokemijskoj industriji. Prema dosadašnjim istraživanjima, genetski modificirani enzim HheA-N178A pokazao je povećanu enantioselektivnost u odnosu na divlji tip HheA te smanjenu regioselektivnost u katalizi azidolize stiren-oksida. 2.Provedena ispitivanja u ovom radu usredotočenasuna određivanje aktivnosti i enantioselektivnosti HheA-N178A na setufluoriranih derivata stiren- oksidakao supstrata uznatrijev azid kao nukleofil(slika1). Racemični supstrati pripravljeni su sintetskim metodama, a enzim HheA-N178A ekspresijom u stanicama E. coli MC1061. Enzimske reakcijeprovedene su u Tris- SO4 puferu koristeći sirovi enzimski ekstrakt. Tijek reakcija praćen je plinskom kromatografijom, a enantiomerna čistoća produkata određena je plinskom ili tekućinskom kromatografijom visoke djelotvornosti koristeći kolone s kiralnom stacionarnom fazom.

Primjenom zelene kemije u proizvodnji biološki aktivnih spojeva nastoji se kemijsku sintezu zamijeniti biokatalizom, a organska otapala s alternativnim zelenim otapalima. U ovom radu ispitana je mogućnost primjene kvasca Saccharomyces cerevisiae kao biokatalizatora za enantioselektivnu redukciju 1-(3, 4- dimetilfenil)etanona i 1-(2, 4, 6- trimetilfenil)etanona u prirodnim eutektičkim otapalima na bazi kolin-klorida. Prirodna eutektična otapala pokazala su se kao povoljan odabir za ispitivane redukcije obzirom na postignuto iskorištenje reakcije te enantiomerni suvišak. Na temelju dobivenih rezultata, zaključuje se da se vrijednosti ispitanih parametara povećavaju povećanjem udjela vode te uporabom supstrata s manjim brojem supstituenata čiji ortho- položaj na fenolnom prstenu nepovoljno utječe na redukciju.

Optically active chiral building blocks are widely used as intermediates in the production of drugs, organic chemicals and agricultural chemicals. As the conventional, environmentally hazardous chemical synthesis is expensive and extremely complicated, there was a rising need to introduce environmentally friendly approaches into the preparation of various commercial products. Herein, two tools of green chemistry were used: performing reactions by biocatalysis and use of alternative green solvents – natural deep eutectic solvents (NADES). For that purpose, due to stereoselectivity of the plant and yeast enzymes, two plant callus cultures - sugar beet and cactus, and baker's yeast Saccharomyces cerevisiae were used as biocatalysts for the asymmetric reduction of 3- methylacetophenone, 3, 4-dimethylacetophenone and 2, 4, 6- trimethylacetophenone into corresponding optically active phenyl-ethanol in choline chloride-based NADES. In summary, we successfully developed an eco- friendly enantioselective reduction in cholinium-based NADES. However, the impact of NADES on whole- cell biocatalysis cannot be simply explained by a single interpretation involving a complicated interaction between them.

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.

Značajan upliv žena na tržište rada znači novu društvenu realnost, obitelj s dva zaposlena partnera. Iako je ulazak u svijet plaćenog rada od iznimne važnosti, ne znači da je posvećenost obitelji jednaka kod žena i muškaraca. Cilj ovog rada je problematizirati poziciju žene kao zaposlene majke, te empirijski ispitati njena iskustva u usklađivanju obiteljskih i radnih obveza. U okviru kvalitativne metodologije proveden je polustrukturirani intervju na uzorku od devet zaposlenih majki. Uvidom u rezultate istraživanja može se zaključiti da sugovornice ističu svoju ulogu i dužnosti majke kao središnje uloge njihovog života. Rezultati istraživanja ukazuju na to da se žene nalaze pod dvostrukim opterećenjem, jer uz svoje odgovornosti u obitelj imaju i obveze na poslu koje moraju izvršiti. Za razliku od svojih također zaposlenih partnera, obavljaju daleko više kućanskih poslova, te je briga o djeci većinom njihova odgovornost, gdje se uočava nejednaka podjela rada unutar obitelji. Bez obzira na pritisak i stres ne bi pristale na odustajanje od zaposlenja van kuće. Iz toga je vidljivo da se sugovornice nalaze u jednom procijepu, vodeći bitku između tradicionalnih zahtjeva „idealne“ majke i supruge koja svo svoje vrijeme posvećuje djeci i obitelji, i želje za nadogradnjom sebe kao žene kroz plaćeni rad i vrijeme za osobne interese. A significant influx of women into labor market introduces a new social reality, consisting of families with two employed partners. The entry into the world of paid work is of utmost importance, but it is not followed by equal amount of commitment to the family by men and women. The objective of this paper is to problematize the position of women as working mothers and empirically examine their experience in reconciling family and work obligations. Semi-structured interview was conducted within qualitative methodology on a sample of nine working mothers After examining the results it was concluded that the interviewees point out as central their role and responsibilities as mothers. The research results indicate that women find themselves under double load because along with their family responsibilities they have their own work responsibilities that they have to perform. Unequal division of labor is noted when unlike their also employed partners, women do far more housework, and childcare. However, regardless the pressure and the stress they might undergo, quiting their paid job is not an option. It is noted that the interviewees are leading a battle between traditional demands of a "perfect" mother and wife who devotes all of her time to her children and family, and the desire to build themselves as women through out paid work and time for their own personal interests.