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Metoda izlaganja heterolognih proteina na površini stanice kvasca Saccharomyces cerevisiae alternativa je klasičnim metodama imobilizacije proteina, a temelji se na fuziji gena koji kodira za protein od interesa sa genom koji kodira za neki od proteina stanične stijenke. Optimizacija sustava za izlaganje proteina na površini stanice kvasca u ovome je radu provedena uvođenjem promjena u sekvencu proteina Pir2 te promjenom pozicije proteina od interesa unutar sekvence proteina Pir2. U promatranom sustavu mjerenjem aktivnosti ˝reporter˝ enzima β-laktamaze te metodom imunoblota proteina stanične stijenke procijenjena je količina rekombinantnog proteina Pir2bla u staničnoj stijenki. Navedenim metodama promatran je i utjecaj mutacija u endocitozi, korištenjem mutanata end3 i end6, na efikasnost izlaganja rekombinantnog proteina na površini stanice u svrhu daljnje optimizacije sustava za imobilizaciju. Surface display of enzymes at the cell wall of yeast Saccharomyces cerevisiae presents an alternative to chemical immobilization methods. Methods for yeast surface display are based on the fusion of a gene encoding a protein of interest with a gene encoding one of the cell wall proteins. Optimization of the protein surface display system in this paper was performed by introducing changes in the Pir2 protein sequence and changing the position of the reporter protein within the Pir2 protein sequence. In the observed system, the amount of recombinant Pir2bla protein in the cell wall was estimated by measuring the activity of the reporter enzyme β-lactamase and the immunoblot of yeast cell wall proteins. Before mentioned methods were also used to investigate the impact of mutations in endocytosis, by using mutants end3 and end6, on efficiency of surface display system in order to optimize immobilization method.

„Yeast surface display“ sustav predstavlja metodu imobilizacije rekombinantnih proteina na površinu stanice kvasca pri čemu se gen koji kodira za željeni protein fuzionira sa čitavim genom ili fragmentom gena koji kodira za jedan od proteina stanične stijenke. Ova genetička fuzija omogućuje ekspresiju i ugradnju rekombinantnog proteina u staničnu stijenku kvasca. Osnovni nedostatci ove metode su relativno mala količina na površinu stanice ugrađenih rekombinantnih proteina, potencijalno smanjenje aktivnost rekombinantnih proteina uslijed fuzije sa proteinom stijenke te ponekad njihova hiperglikozilacija. Glikozilacija je postsintetska preinaka proteina koja može utjecati na smatanje, aktivnost i stabilnost proteina. Cilj ovog rada jest ispitati utjecaj O-glikozilacije na efikasnost ugradnje i aktivnost rekombinantnog enzima β-laktamaze u staničnu stijenku. Gen koji kodira za ovaj enzim je u jednom slučaju fuzioniran sa genom koji kodira za Pir2, a u drugom slučaju sa dijelom gena koji kodira za Ccw12 protein stijenke. Rekombinantni enzim eksprimiran je u stanicama kvasca divljeg tipa i u stanicama koji imaju mutaciju u O-glikozilaciji proteina (pmt mutanti). Količina enzima koja se ugradila u stijenku analizirana je mjerenjem aktivnosti enzima korištenjem nitrocefina kao supstrata i western blot analizom. Yeast „surface display“ system represents an alternative method of immobilization of recombinant proteins on the yeast cell wall surface in which the gene encoding for the protein of interest is fused with whole or with fragment of a gene encoding for a native yeast cell wall proteins. This genetic fusion provides constant expression and incorporation of the recombinant protein into the cell wall. However, one of the major disadvantages of this method are relatively low yield of the recombinant protein in the cell wall, potential low activity of incorporated recombinant protein due to their fusion with native cell wall protein and their hypermannosylation. Glycosylation is one of major posttranslational modifications which effects protein folding, their activity and stability. Theme of this thesis is to study the effect of O-glycosylation on incorporation of the recombinant enzyme β-lactamase into the cell wall. In one case, the gene encoding for this enzyme is fused with gene encoding for Pir2 protein, and in the other the gene is fused with the gene encoding for Ccw12 protein. The recombinant enzyme was expressed in the wild type yeast cells and in O-glycosylation mutants (pmt mutants). The amount of enzyme in the cell wall is assessed by measuring β-lactamase activity using nitrocefine as substrate and by western blot.

Izlaganje proteina na staničnoj površini (˝surface display˝) predstavlja poboljšanu tehniku imobilizacije proteina te omogućuje kontinuiranu sintezu rekombinantnih proteina zahvaljujući fuziji gena koji kodira za ispitivani enzim i gena koji kodiraju za nativne proteine stanične stijenke. U ovom radu rekombinantni protein β-laktamaza (gen bla) fuzioniran je sa proteinom Pir2, odnosno sa dijelom proteina Ccw12. Pir2 veže se za stijenku svojim Nterminalnim dijelom. Dio gena CCW12, korišten u ovom radu, kodira za C-terminalnu signalnu sekvencu na koju se veže GPI-sidro, preko kojeg se protein veže u stijenku. Najveći je nedostatak ovakvog pristupa mali prinos imobiliziranih proteina u stijenku. S pretpostavkom da mutacije u N-glikozilaciji i endocitozi povećavaju učinkovitost imobilizacije ispitivan je utjecaj mutacija u N-glikozilaciji (och1 mutant i mnn mutanti) te mutacija u endocitozi (vam4 mutant i end mutanti) na efikasnost ugradnje rekombinantnog proteina u stijenku mjerenjem aktivnost β-laktamaze, što omogućuje procjenu količine imobiliziranog rekombinantnog proteina u staničnoj stijenci. „Surface display“ system provides enhanced immobilization technique that enables countinuous synthesis of recombinant protein. This is possible because of the genetic fusion between genes encoding for native cell wall proteins and enzyme that is reasearched. In this study the enzyme β-lactamase (gene bla) and Pir2 or a fragment of Ccw12 are fuzed. Pir2 is linked to the cell wall on its N-terminal end, while Ccw12 is linked to the cell wall by GPI anchor on C- terminal region. One of the major disadvantages of this approach is low yield in protein immobilization. With the assumption that mutation in N-glycosylation and endocytosis enhances surface display system efficiency, this system was examined in mutants in Nglycosylation (och1 mutant and mnn mutants) and in mutants in endocytosis (vam4 mutant and end mutants). By measuring β-lactamase activity, it is possible to estimate the amount of recombinant protein immobilized in cell wall.

Scw4 protein u stijenku kvasca Saccharomyces cerevisiae veže se kovalentnim i neokovalentnim vezama, međutim, točan mehanizam kovalentnog povezivanja i dalje je nepoznat. Polazišna je pretpostavka da je način vezanja sličan onome PIR proteina, s obzirom na jednak način ekstrakcije kovalentno vezane frakcije ovog proteina iz stijenke. Cilj ovog istraživanja bio je odrediti koja je aminokiselina odgovorna za kovalentno vezanje Scw4 u stijenku kvasca. U ciljanu regiju gena SCW4 uvedene su mutacije, a uzgoj mutanata proveden je u nepuferiranom mediju, te pri pH=4 i pH=7. Nakon uzgoja nekovalentno vezani proteini su izolirani iz stijenki pomoću vrućeg SDS-a, a kovaletno vezani pomoću 30 mM NaOH. Dobiveni proteinski ekstrakti analizirani su Western blot metodom. Iz dobivenih rezultata možemo zaključiti kako je za kovalentno vezanje Scw4 u stijenku S. cerevisiae odgovoran ostatak glutamina Q₂₄₁. The Scw4 protein of Saccharomyces cerevisiae yeast can be bound both covalently and non-covalently to the cell wall, however, the exact mechanism of covalent binding is still unknown. The initial assumption is that the binding mechanism is similar to that of PIR proteins, given the same method of extraction of covalently bound fraction of these proteins from the yeast cell wall. The aim of this study was to determine which amino acid is responsible for covalent binding of Scw4 to the yeast wall. Mutations were introduced into the target region of the SCW4 gene, followed by the cultivation in an unbuffered medium, as well as at pH=4 and pH=7. After cultivation, non-covalently bound proteins were isolated by hot SDS, while covalently bound proteins were extracted by 30 mM NaOH. Protein extracts were analyzed by the Western blot method. The obtained results showed that glutamine Q₂₄₁ is responsible for covalent binding of Scw4 to the cell wall of S. cerevisiae.

U novije vrijeme, veliki interes za heterologne proteine dovodi do potrebe za pronalaskom efikasnih tehnika za njihovu proizvodnju. Tehnika izlaganja heterologih proteina na površini stanične stijenke kvasca je značajan predmet istraživanja u znanosti i industriji. Njezinom primjenom omogućava se istovremena proizvodnja i sekrecija heterolognih proteina te njihova imobilizacija na površini stanice i primijena kvasaca kao staničnih biokatalizatora. Osnovni nedostatak metode je relativno niska koncentracija imobiliziranih proteina na površini stanica. Ta je činjenica glavni razlog sve većeg broja istraživanja u svrhu poboljšanja metode. Nastoje se pronaći optimalni sustavi za izlaganje heterolognih proteina te pogodni domaćini. Uz do sada najviše istraženi kvasac S. cerevisiae, sve veća važnost se pridaje metilotrofnim kvascima. Cilj ovog rada je prikazati nekonvencionalne metilotrofne kvasce, P. pastoris i H. polymorpha, kao pogodne domaćine za proizvodnju heterolognih proteina te njihovo izlaganje na površini stanica. In recent times, the great interest in heterologous proteins has led to the need of finding efficient techniques for their production. Yeast surface display of heterologous proteins has become a significant subject of research in science and industry. Its application enables the simultaneous production and secretion of heterologous proteins, as well as their immobilization on the cell surface and the use of yeast as whole-cell biocatalysts. The main disadvantage of the method is the relatively low concentration of immobilized proteins on the cell surface. This fact is the main reason for the progress of research in order to improve the method. Great effort has been put into finding the optimal systems for surface display of heterologous proteins and finding the suitable host. In addition to the most researched yeast S. cerevisiae so far, increasing importance is given to methylotrophic yeast. The aim of this study is to present non-conventional methylotrophic yeasts, P. pastoris and H. polymorpha, as suitable hosts for the production of heterologous proteins and their display on the cell surface.

Protein Scw4 u staničnu stijenku kvasca Saccharomyces cerevisiae može se vezati nekovalentno i kovalentno, no i dalje nije poznato koji dio proteina je odgovoran za kovalentno povezivanje. U prethodnom istraživanju uvedena je mutacija u jednu od regija koja je slična Pir repetitivnim regijama za koje se pretpostavlja da bi mogle biti odgovorne za kovalentno povezivanje. Također je utvrđeno kako se nativni Scw4 u staničnoj stijenci pojavljuje u tri forme različite veličine – neprocesiranoj, procesiranoj sa Kex2 proteazom te procesiranoj sa japsinskim proteazama. U ovom radu ispitivan je način vezanja mutiranog proteina Scw4 u staničnu stijenku kvasca Saccharomyces cerevisiae pri uzgoju na pH 4 i pH 7. Prethodno izraĎen konstrukt sa mutacijom umnožen je u bakteriji Escherichia coli, izoliran te su njime transformirane kvaščeve stanice. Kvasci su uzgajani u selektivnoj podlozi za uzgoj u uvjetima indukcije GAL promotora. Nakon uzgoja provedena je izolacija i analiza načina vezanja proteina stanične stijenke metodama SDS elektroforeze te metodom western blota. Na osnovu dobivenih rezultata može se zaključiti da je ispitivana regija u sekvenci Scw4 proteina bitna za njegovo kovalentno vezanje u staničnu stijenku. Protein Scw4 can be bound into the cell wall of yeast Saccharomyces cerevisiae non-covalently and covalently. However, the part of the protein responsible for the covalent bond remains unknown. According to previous research, a mutation has been introduced into one of the regions similar to Pir repetitive regions which are supposed to be responsible for the covalent bonding. It has also been established that the Scw4 protein can appear in the cell wall in three different sized forms – unprocessed, processed with Kex2 protease, and processed with yapsin proteases. In this particular research, the way of binding mutated Scw4 protein grown on pH 4 and pH 7 has been examined. A previously made mutated construct has been replicated in Escherichia coli, isolated, and used to transform yeast cells which have been grown on selective media under the induction of the promotor. Cell wall proteins have been isolated and analysed by SDS PAGE and by western blotting. According to the results obtained, it can be concluded that the examined region in the Scw4 protein sequence is important for its covalent binding to the cell wall.

Pichia pastoris često je korišteni mikroorganizam u biotehnološkoj proizvodnji heterolognih proteina. Zbog mogućnosti rasta do visokih staničnih gustoća važna je mogućnost njegove primjene u dobivanju kemikalija na ekološki prihvatljiv i ekonomski isplativ način. U ovom radu konstruiran je plazmidni vektor pogodan za transformaciju kvasca Pichia pastoris koji nosi gen za rekombinantni protein Ccw12XR iz kvasca Saccharomyces cerevisiae. Rekombinantni gen se sastoji od dijela koji kodira za enzim ksiloza reduktazu (XR) i dijela koji kodira za protein Ccw12 stanične stijenke kvasca S. cerevisiae. Rekombinantni gen umnožen je PCR metodom i ugrađen TA ligacijom u plazmidni vektor pGEM®-T Easy. Plazmid je umnožen u kompetentnim stanicama E. coli, nakon čega je iz plazmida izrezan rekombinantni gen CCW12XR korištenjem SapI restrikcijskog enzima. Naposljetku je CCW12XR gen ligiran s lineariziranim plazmidom pBSY3Z. Uspješnost ligacije je, nakon umnažanja plazmidnog vektora u kompetentnim stanicama E. coli, provjerena restrikcijom pomoću odabranih restrikcijskih enzima i elektroforezom na agaroznom gelu. Pichia pastoris is a commonly used host in the biotechnological production of heterologous proteins. Since it can grow to very high cell densities, Pichia pastoris is applicable in the eco-friendly and economical production of chemicals. This thesis describes the construction of the shuttle vector, suitable for transformation of Pichia pastoris carrying the gene for the Ccw12XR recombinant protein from Saccharomyces cerevisiae. The recombinant gene encodes both the xylose reductase (XR) and the Ccw12 cell wall protein of S. cerevisiae. The recombinant gene is amplified using PCR and ligated into pGEM®-T Easy. The plasmid is replicated in competent Escherichia coli cells, following which the recombinant CCW12XR gene is restricted from the constructed plasmid using SapI. The isolated CCW12XR gene is then ligated with the linearized pBSY3Z plasmid. Finally, following the replication of the shuttle vector in component E. coli cells, the ligation efficiency is verified via restriction analysis and DNA electrophoresis.

Kvasac Saccharomyces cerevisiae jedan je od najčešće korištenih mikroorganizama u biotehnologiji, biomedicini i raznim znanstvenim istraživanjima. U ovom radu kvasac se koristio za ekspresiju enzima za razgradnju atrazina koji potječu iz Pseudomonas sp. ADP. Atrazin je herbicid koji je zabranjen od strane EU jer zbog svog kumulativnog efekta izaziva onečišćenje okoliša. Metabolički put razgradnje atrazina se sastoji od šest enzimskih reakcija od kojih je svaka katalizirana s po jednim enzimom. Cilj rada je bio eksprimirati i imobilizirati na površini stanične stijenke kvasca S. cerevisiae prva četiri enzima metaboličkog puta razgradnje atrazina (AtzA, AtzB, AtzC i AtzD), a u tu svrhu korišteni su proteini stanične stijenke kvasca Pir4 i Ccw12 s kojima su ti enzimi bili genetički fuzionirani. Uspješnost ekspresije i imobilizacije rekombinantih proteina provjeravala se Western blot analizom proteina ekstrahiranih iz izoliranih staničnih stijenki kvasca. Osim toga ispitivala se i aktivnost eksprimiranih rekombinantih enzima u tekućem mediju i na krutoj podlozi u Petrijevim zdjelicama. Yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms in biotechnology, biomedicine and various scientific research. In this thesis yeast was used to express the atrazine degradation enzymes originated from Pseudomonas sp. ADP. Atrazine is a herbicide that is banned from the EU because of its cumulative effect on environmental pollution. The metabolism of atrazine degradation consists of six enzymatic reactions each of which is catalysed with one enzyme. The aim of this study was to express and immobilize the first four enzymes of the atrazine degradation pathway (AtzA, AtzB, AtzC and AtzD) on the surface of the S. cerevisiae cell. For this purpose, Pir4 and Ccw12 yeast cell wall proteins were genetically fused with these enzymes. Success of expression and immobilization of the recombinant proteins was verified by Western blot analysis of proteins extracted from isolated yeast cell walls. In addition, the activity of expressed recombinant enzymes in the liquid medium and on the solid substrate in Petri dishes was also investigated.

Pichia pastoris je metilotrofni kvasac koji se upotrebljava kao uspješni sistem za ekspresiju heterolognih proteina. U ovom radu, P. pastoris je upotrijebljena za ekspresiju heterolognog rekombinantnog proteina ksiloza reduktaze na površini stanične stijenke kvasca. Ksiloza reduktaza je enzim koji katalizira oksidoredukcijsku reakciju u kojoj se ksiloza reducira u ksilitol uz oksidacije NADPH u NADP+. Ksilitol je nisko kalorični šećerni alkohol koji se primjenjuje u prehrambenoj, farmaceutskoj i kozmetičkoj industriji. Biotehnološka proizvodnja ksilitola predstavlja ekonomičnije riješenje u odnosu na kemijsku proizvodnju u industriji. U ovom radu P. pastoris je transformirana plazmidom pBSY3ZCcw12XR koji nosi gen za rekombinantnu ksiloza reduktazu, ispitana je uspješnost ekspresije i ugradnje rekombinantnog enzima u staničnu stijenku kvasca, te aktivnost imobiliziranog enzima. Pichia pastoris is a methylotrophic yeast that is used as a successful heterologous protein expression system. In this experiment, P. pastoris was used for the expression of the heterologous xylose reductase recombinant protein on the surface of the cell wall. Xylose reductase is an enzyme that catalyzes the oxidoreduction reaction in which xylose is reduced to xylitol along with the oxidation of NADPH to NADP+. Xylitol is a low calorie sugar alcohol that is used in the food, pharmaceutical and cosmetic industries. Biotechnological production of xylitol represents a more economical solution compared to chemical production. In this experiment, P. pastoris was transformed with the plasmid pBSY3ZCcw12XR carrying the gene for recombinant xylose reductase. Along with examining the expression and immobilization of the recombinant enzyme, the activity of xylose reductase was also tested.

Stanična stijenka kvasca Saccharomyces cerevisiae je ekstracelularna organela građena od polisaharida i proteina koja stanici pruža oblik, čvrstoću, mehaničku zaštitu, osmotsku stabilnost, ali služi i u komunikaciji s drugim stanicama. Proteini sudjeluju u održavanju strukture stanične stijenke i komunikaciji stanica s okolinom, a osim prema funkciji razlikuju se prema građi i načinu vezanja u staničnu stijenku. Poznato je da se Ccw5 u staničnu stijenku veže esterskom vezom između β-1,3-glukana i ostatka glutamina unutar tzv. repetitivne sekvence ovog proteina. Međutim, objavljeno je nekoliko radova u kojima autori tvrde da se Ccw5 u stijenku kovalentno veže disulfidnim mostovima. Stoga je u ovom radu ispitana uloga disulfidnih mostova u ugradnji proteina Ccw5. Dobiveni rezultati su potvrdili da disulfidni mostovi nemaju ulogu u vezanju Ccw5, već da je on vezan esterski u stijenku, jer i nakon višestrukog tretmana sa SDS-om i/ili β-merkaptoetanolom u stijenci zaostaje kovalentno vezana frakcija ovog proteina koja se ekstrahira pomoću NaOH. Saccharomyces cerevisiae cell wall is an extracellular organelle built from polysaccharides and proteins, which gives the cell its shape, firmness, protection, osmotic stability and participates in communication with other cells. Proteins maintain the structure of cell wall and are used for communication with surroundings. They vary in function and in the type of bond by which they are linked to the cell wall. It is known that Ccw5 is linked to the cell wall by ester linkage between glutamine from repeated sequence of protein and β-1, 3-glucan. However, several articles were published stating that Ccw5 is linked to the cell wall covalently by disulphide bonds. Therefore, the aim of this work was to examine the role of disulphide bonds in incorporation of Ccw5. Obtained results confirmed that disulphide bonds don't have a role in linking of Ccw5, and that Ccw5 is linked to the cell wall by ester linkage, because even after multiple treatment of cell walls with SDS and/or β-mercaptoethanol, covalently linked fraction of Ccw5 remains linked to cell wall and is extracted by NaOH.

Kloniranje gena ili dijelova gena u molekularnoj biologiji je skup metoda kojima se željene sekvence iz DNA molekule umnažaju u stanici domaćinu. Eksperimentalni rad započinje umnažanjem željenog fragmenta DNA konvencionalnim metodama poput PCR-a i cijepanjem DNA restrikcijskim enzimima. Nakon toga slijedi ligacija fragmenta DNA (inserta) i lineariziranog plazmidnog vektora kako bi se osiguralo nastajanje plazmida koji se može replicirati u stanici domaćinu te unošenje plazmida u stanicu domaćina u kojoj dolazi do njegovog umnažanja. Na taj način iz jedne rekombinantne DNA nastaje veliki broj istovjetnih molekula DNA što predstavlja samu bit kloniranja. U ovom radu konstruiran je insert Δ246 iz gena SCW4 kvasca Saccharomyces cerevisiae te je ugrađen takozvanom TA-ligacijom u plazmidni vektor pGem-T Easy. Takvim plazmidom su transformirane kompetentne stanice bakterije Escherichia coli. Naposljetku je iz transformiranih stanica izoliran umnoženi plazmid, koji je podvrgnut restrikcijskoj analizi odgovarajućim restrikcijskim enzimima te je rezultat provjeren elektroforetskom analizom restrikcijske smjese. Cloning of genes or gene fragments in molecular biology implies set of methods used to construct desired sequences of DNA and replicate them in host cells. Experimental work primarily sets on creating fragments of DNA by conventional methods including PCR and enzymatic digestion of obtained DNA fragments. Thereafter follows ligation of DNA fragment and linearised vector to ensure formation of plasmid which can replicate itself in the host cell. In this way, out of one recombinant DNA a great number of copies are produced, what is the main goal of cloning. This work is based on the construction of DNA fragment of yeast's Saccharomyces cerevisiae gene SCW4 and it`s ligation into the plasmid vector pGem-T Easy by TA-ligation. This newly created plasmid was introduced into competent cells of Escherichia coli. Cloned plasmid was isolated from transformed cells and the outcome was verified through restriction analisys.

Stanična stijenka kvasca je čvrsta i elastična ekstracelularna struktura građena od polisaharida i proteina. Proteini stijenke međusobno se razlikuju po svojoj funkciji i po načinu ugradnje u staničnu stijenku. Scw4 je protein koji se u stijenku djelomično ugrađuje nekovalentnim, a djelomično kovalentnim vezama. Smatra se da je način kovalentnog vezanja Scw4 u stijenku jednak načinu vezanja proteina PIR porodice, odnosno vezanje esterskom vezom labilnom u alkalnim uvjetima. Prema nekim autorima PIR proteini se u stijenku kvasca ugrađuju disulfidnim mostovima, stoga je u ovom radu ispitano imaju li disulfidni mostovi ulogu u ugradnji kovalentno vezane frakcije Scw4 u stijenku. Osim toga ispitano je ima li β-merkaptoetanol ulogu u ekstrakciji nekovalentno vezane frakcije Scw4. Tretman SDS-om sa ili bez β-merkaptoetanola pokazao je da glavnu ulogu u ekstrakciji nekovalentno vezane frakcije proteina Scw4 ima SDS. Neuspješna ekstrakcija sa β-merkaptoetanolom pokazala je da se Scw4 kovalentno u stijenku ne ugrađuje disulfidnim mostova. The yeast cell wall is firm and flexible extracellular structure constructed of polysaccharide moieties and mannoproteins. Except for their physiological role cell wall proteins differ according to the way they are linked to glucan. It is known that Scw4 is partly bound to the cell wall covalently and partly non-covalently. Covalently linked Scw4 is most probably attached to the cell wall in the same way as PIR proteins, by alkali-labile ester linkage. However, some authors think that PIR proteins are linked to the cell wall by disulphide bonds. Therefore, role of disulphide bonds in the integration of Scw4 in the cell wall was examined in this work. Furthermore, some experiments were conducted to find out if β-mercaptoethanol has a role in the extraction of non-covalently linked Scw4. According to the results, non-covalently linked Scw4 is SDS-extractable regardless of the presence of β-mercaptoethanol which means that the SDS has main role in the extraction of non-covalently linked Scw4. Scw4 is covalently linked most probably by ester linkage because it is NaOH extractable, and unsuccessful extraction with β-mercaptoethanol shows that disulphide bonds doesn't have role in the integration of Scw4 in the cell wall.

Saccharomyces cerevisiae jedan je od najčešće korištenih mikroorganizama u biotehnološkoj proizvodnji, pa se tako koristi i u proizvodnji ksilitola, prirodnog zaslađivača s velikim prehrambenim prednostima. Enzim odgovoran za mikrobiološku proizvodnju ksilitola, ksiloza reduktaza, katalizira reakciju redukcije ksiloze u ksilitol. Ksiloza reduktaza prirodno je lokalizirana u citoplazmi stanice kvasca što znači da izolacija finalnog proizvoda uključuje razbijanje stanice i više koraka pročišćavanja. Zbog relativno malog prinosa i kompleksnog pročišćavanja ksilitol na tržištu ima visoku cijenu. Tema ovog rada jest ekspresija i imobilizacija heterologne rekombinantne ksiloza reduktaze na površini stanice kvasca čime bi se izolacija proizvoda olakšala, a cijena konačnog proizvoda snizila. U tu je svrhu iskorišten protein Pir4, protein stanične stijenke kvasca, s kojim je ksiloza reduktaza fuzionirana i preko kojeg je vezana na staničnu stijenku. U radu je ispitana uspješnost ekspresije i ugradnje u stijenku te aktivnost tako imobiliziranog rekombinantnog enzima. Saccharomyces cerevisiae is one of the most used microorganisms in biotechnological production, so it's also used in production of xylitol, natural sweetener with many nutritional advantages. Enzyme responsible for microbiological production of xylitol, xylose reductase, catalyses the reduction reaction of xylose to xylitol. Xylose reductase is naturally localized in yeast cell cytoplasm meaning that the isolation of the final product includes breaking the yeast cell and several steps of purification. Because of low yield and complexity of purification, xylitol has high price on the market. Theme of this thesis is expression and immobilization of heterologous recombinant xylose reductase on the surface of yeast cell meaning that the isolation of product would be easier and the price lower. For that purpose yeast cell wall protein Pir4is fused with xylose reductase serving as anchor through which xylose reductase binds to the yeast cell wall. In this thesis, the efficacy of expression and incorporation of the immobilized recombinant enzyme to the cell wall is examined, as well as its activity.

Scw4 je protein koji se u staničnoj stijenci kvasca Saccharomyces cerevisiae veže kovalentno i nekovalentno, ali nije poznato koji dio gena kodira za regiju koja mu omogućuje kovalentno povezivanje. Provedeno je ispitivanje načina vezanja u stijenku nakon delecije dijelova gena SCW4 koji kodiraju za regije za koje se pretpostavlja da bi mogle omogućiti kovalentno povezivanje. U tu je svrhu kvasac uzgojen u selektivnoj YNB podlozi nakon čega su izolirane stijenke i njihovi proteini kuhanjem u SDS-u uz dodatak β-merkaptoetanola odnosno tretmanom stijenki s 30 mM NaOH. Izolirani proteini su zatim analizirani SDS elektroforezom po Laemmli-u i metodom Western blot. Prema dobivenim rezultatima može se pretpostaviti da se Scw4 protein u staničnu stijenku kvasca veže kovalentno preko regije koja je prema broju i redoslijedu glutaminskih ostataka slična repetitivnoj sekvenci PIR proteina. Dobivene rezultate je potrebno potvrditi daljnjim istraživanjima. Scw4 is a protein which can be bound covalently and non-covalently into the cell wall of yeast Saccharomyces cerevisiae, but it is not known which region of protein allows its covalent linkage. The way of its binding was examined after deletion of SCW4 gene segments encoding regions that could be responsible for covalent attachment of Scw4. For this purpose, the yeast was cultivated in a selective YNB medium and after that, the cell walls and proteins were isolated by boiling in SDS with β-mercaptoethanol or by treatment with a 30 mM NaOH. Isolated proteins were examined by SDS electrophoresis according to Laemmli and by Western blotting. According to the results, it can be assumed that the protein Scw4 is bound covalently via a region that has an arrangement and number of glutamine residues similar to that found in the repetitive sequence of PIR proteins that is responsible for its covalent binding. This results should be confirmed in further experiments.