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Protein immobilization is of utmost importance in many areas, where various proteins are used for selective detection of target compounds. Despite the importance given to determine the amount of immobilized protein, there is no simple method that allows direct, noninvasive detection. In this work, a method based on pH transition, occurring during change of solution ionic strength, was developed. The method utilized the ionic character of the immobilized protein while implementing biologically compatible buffers. Five different proteins, namely, glucose oxidase, horseradish peroxidase, bovine serum albumin, lysozyme, and protein A, were immobilized in different amounts on a porous polymeric matrix, and their pH transition was measured using lactate buffer of various concentrations and pH values. A linear correlation was found between the amount of immobilized protein and the amplitude of the pH transition, allowing the detection down to 2 nmol of immobilized protein. By changing the buffer concentration and pH, the sensitivity of the method could be tailored. Criteria based on the symmetry of the pH transition peak have been developed to determine if a particular measurement is within a linear range. In addition, a mathematical model was developed enabling prediction of pH transition profiles based solely on the protein amino acid sequence, the buffer pK$_a$ value(s), and the amount of immobilized protein. Hence, it can be used to design pH transition method experiments to achieve the required sensitivity for a target sample. Since the proposed method is noninvasive, it can be routinely applied during optimization of the immobilization protocol, for quality control, and also as an in-process monitoring tool.

Uvod: Zgornja okončina je najaktivnejši in najmanj zaščiteni del telesa, ki opravlja številne funkcije. Posledično poškodbe zgornje okončine predstavljajo največji delež med vsemi poškodbami. Ob poškodbi je pomembno, da v okviru prve pomoči s pravilno imobilizacijo zagotovimo ustrezno oporo poškodovanemu udu, dokler poškodovanec ne dobi ustrezne strokovne oskrbe. Namen: Namen magistrskega dela je bil ugotoviti, kakšno je znanje dajalcev prve pomoči (laikov) v zvezi z oskrbo dejanskih poškodb kosti in sklepov zgornje okončine. Metode: Uporabili smo kvalitativno metodo dela – analizo slik. Predhodno smo v uvodnem delu uporabili metodo kompilacije s povzemanjem znanj, stališč in mnenj slovenskih in tujih avtorjev o obravnavni tematiki. Za analizo slik smo kot merski instrument izdelali ocenjevalni list, ki je sledil zasnovi ocenjevanja oskrbe poškodb na preverjanjih usposobljenosti ekip prve pomoči in je prirejen glede na aktualne smernice za imobilizacijo zgornje okončine. Drugi sklop podatkov smo zbrali s pomočjo namensko oblikovanega anonimnega vprašalnika za poškodovance. Rezultati: Vzorec je zajemal 50 poškodovancev, pregledanih v Urgentnem kirurškem bloku Univerzitetnega kliničnega centra v Ljubljani. Povprečno število točk, ki so jih po naših merilih dosegli dajalci prve pomoči pri izvedeni imobilizaciji (M = 3,68 točke SD = 1,671 točke), je pod srednjo vrednostjo ocenjevalne lestvice. Statistično pomembne razlike smo ugotovili v subjektivni oceni bolečine poškodovancev na vizualni analogni skali pred imobilizacijo in po njej (Z = -4,937 p < 0,001 < 0,05). Osebe, ki so pogosteje v stiku z vsebinami prve pomoči, so bolje oskrbele poškodovano okončino kot laiki, ki tega stika nimajo (MW = 166 p < 0,001 < 0,05). Razprava in zaključek: Spodbudno je, da dajalci prve pomoči uporabljajo za imobilizacijo zgornje okončine enostavne pripomočke iz kompleta prve pomoči. Glede na postavljena merila so pri oceni oskrbe poškodb kosti zgornje okončine dosegli podpovprečno število točk. Hkrati smo ugotovili, da se je bolečina pri večini poškodovancev po imobilizaciji glede na izhodiščno stanje zmanjšala. Na osnovi tega zaključujemo, da je v primeru poškodb kosti zgornje okončine bolje narediti povprečno imobilizacijo, kot da imobilizacije sploh ne naredimo. Osebe, ki so bolj v stiku z vsebinami prve pomoči, naredijo boljšo imobilizacijo, kar je pričakovano. Na tečajih prve pomoči je treba laike usposobiti za izvedbo kakovostne imobilizacije. Introduction: The upper extremities are the most active and the least protected body parts, performing many functions. As a result, upper limbs account for the largest share of all injuries. In the event of an injury, it's important that first aid immobilization is done correctly and provides adequate support to the injured limb until the injured person receives appropriate professional care. Purpose: The master's thesis aimed to determine the knowledge first aiders (non-specialists without a medical background) have regarding first aid for upper limb bone and joint injuries. Methods: We used a qualitative method – image analysis. In the introductory part, we used the compilation method to summarize Slovenian and foreign authors' knowledge, points of view, and opinions on the topic. For the image analysis, we developed a scorecard. It followed the concept of care assessment of injuries in first aid competency tests and was adapted to the current guidelines for upper limb immobilization. We collected the second dataset using a purpose-designed anonymous questionnaire for injured people. Results: The sample consisted of 50 injured patients examined in the Emergency Surgical Block of the University Medical Centre in Ljubljana. The average number of points achieved by first responders for the immobilization (M = 3.68 points SD = 1.671 points) is below the median value. Statistically significant differences were found in the subjective assessment of injured patients' pain based on a visual analog scale before and after immobilization (Z = -4.937 p < 0.001 < 0.05). People who are more often in contact with the first-aid content provided better care for injured limbs than people who are not (MW = 166 p < 0.001 < 0.05). Discussion and conclusion: It is encouraging that first aiders use simple tools from a first aid kit to immobilize upper limbs. They have achieved below-average scores according to upper limb bone injury care assessment. At the same time, we found that the pain of most injured patients decreased from baseline after the immobilization. This led to the conclusion that for upper limb bone injuries, average immobilization is better than no immobilization. As expected, people with more knowledge about first aid do a better job of immobilization. First aid courses should train non-specialists to perform proper and good-quality immobilization.

The environmental properties of three geotechnical composites made by recycling wastes were investigated on a laboratory scale and in the field with the use of lysimeters designated for the revitalization of degraded mining sites. Composites were prepared by combining the mine waste with paper-mill sludge and foundry sand (Composite 1), with digestate from municipal waste and paper ash (Composite 2), and with coal ash, foundry slag and waste incineration bottom ash (Composite 3). The results of laboratory leaching tests proved that Composites 1 and 3 are envi- ronmentally acceptable, according to the legislative limits, as the potentially hazardous substances were immobilized, while in Composite 2, the legislative limits were exceeded. In the field lysimeters, the lowest rate of leaching was determined for optimally compacted Composites 1 and 3, while for Composite 2 the leaching of Cu was high. This study proved that optimally installed Composites 1 and 3 are environmentally acceptable for use in construction as an alternative to virgin materials, for the revitalization of degraded mining sites or, along with Composite 2, for closure operations with landfills. In this way, locally available waste streams are valorised and channelized into a beneficial and sustainable recycling practice.

Glavni izzivi biokatalitskih procesov so razvoj zelo aktivnih, robustnih in stabilnih biokatalizatorjev, učinkovita regeneracija in preprečevanje deaktivacije biokatalizatorja. Magnetni delci predstavljajo prilagodljiv nosilec za imobilizacijo encimov ali celic, saj imajo veliko specifično površino z možnostjo funkcionalizacije in omogočajo enostavno imobilizacijo biokatalizatorja z zunanjim magnetnim poljem. V pričujočem delu smo razvili mikropretočno napravo, v kateri smo učinkovito imobilizirali bakterijske spore s pomočjo magnetnih delcev. Preučili smo vpliv pH in različnih magnetnih delcev na lakazno aktivnost spor. Prav tako smo preučili vpliv pH in različnega razmerja med količino magnetnih delcev in spor na uspešnost imobilizacije spor v magnetnem polju. Pri optimalnih pogojih, ki so bili pH imobilizacije 2,5, magnetni delci MagP-NH2 in masno razmerje magnetnih delcev in spor 2,2, je bil izkoristek imobilizacije v pretočnem sistemu 97 %, zadržana lakazna aktivnost spor z magnetnimi delci MagP-NH2 v šaržnem sistemu pri pH 4 pa 88 %. Mikroreaktor z imobiliziranimi sporami v magnetnem polju smo uporabili za oksidacijo 2,2'-azino-bis(3-etilbenzotiazolin)-6-sulfonske kisline. Med potekom pretočnih reakcij smo ugotovili, da se v 5 urah procesa v reaktorju zadrži več kot 83 % spor. Koncentracijo produkta na izstopu iz mikroreaktorja smo spremljali izven sistema z UV/VIS spektrofotometrom, kot tudi z integriranim miniaturiziranim pretočnim spektrofotometrom, ki omogoča pridobivanje podatkov v realnem času. The main challenges of biocatalytic processes are the development of highly active, robust and stable biocatalysts, efficient regeneration and prevention of biocatalyst deactivation. Magnetic particles represent a flexible carrier for the immobilization of enzymes or cells, as they have a large specific surface area with the possibility of functionalization and enable easy immobilization of the biocatalyst with an external magnetic field. In the present work, we developed a microfluidic device in which we effectively immobilized bacterial spores with the help of magnetic particles. We examined the influence of pH and different magnetic particles on the laccase activity of spores. We also studied the effect of pH and different ratios between the amount of magnetic particles and spores on the immobilization efficiency in a magnetic field. At the optimal conditions, which were immobilization pH 2,5, MagP-NH2 magnetic particles and the mass ratio of magnetic particles to spores 2,2, the immobilization efficiency in the flow system was 97 %, the retained laccase activity of spores with MagP-NH2 magnetic particles was 88 % for the batch system at pH 4. A microreactor with immobilized spores in a magnetic field was used for the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid. During the flow reactions, we found that more than 83 % of the spores are retained in the reactor within 5 hours of the process. The concentration of the product at the outlet of the microreactor was monitored outside the system with a UV/VIS spectrophotometer, as well as with an integrated miniaturized flow spectrophotometer, which enables data acquisition in real time.

U ovom radu ispitana je mogućnost pripreme novih biosorbensa imobilizacijom inaktivne biomase gljive Fomitopsis pinicola uklapanjem u kalcijev alginat. Novi biosorbensi su pripremljeni ukapljanjem inaktivne biomase gljive u 1 % i 2 % kalcijev alginat, pri čemu je biomase gljive dodana u različitim količinama u odnosu na masu natrijevog alginata korištenog u postupku imobilizacije (10 %, 20 % i 30 % gljive na masu alginata te u omjerima 1:1, 1:1,5 i 1:2 u odnosu na masu natrijevog alginata). Dobiveni biosorbensi su osušeni te su prije i nakon sušenja određivani promjer i udio vlage te stupanj bubrenja mikrosfera biosorbensa. Nadalje, ispitana je učinkovitost uklanjanja sintetskog bojila kongo crvenila iz vodenih otopina korištenjem dobivenih biosorbensa. Rezultati su pokazali kako je dodatak inaktivne biomase gljive utjecao na povećanje promjera te smanjenje udjela vode dobivenih mikrosfera biosorbensa u odnosu na mikrosfere biosorbensa bez dodatka biomase gljive. Svi biosorbensi pokazali su se učinkovitima za uklanjanje kongo crvenila (i do 80 %) iz vode pri odabranom vremenu kontakta od 6 h. Biosorbensi sa smanjenim udjelom natrijeva alginata (1 %) te povećanim udjelom biomase gljive također su se pokazali učinkovitima za uklanjanje kongo crvenila, ali nije postignuto očekivano povećanje učinkovitosti u odnosu na uzorak bez dodatka biomase gljive. Rezultati dobiveni ovim istraživanjem upućuju na mogućnost upotrebe otpadne inaktivne gljive Fomitopsis pinicola za pripremu biosorbensa, ali i na potrebu provođenja daljnjih istraživanja koja će uključivati detaljniju fizikalno-kemijsku i morfološku karakterizaciju biosorbensa te opsežnije biosorpcijske eksperimente. In this study, the possibility of preparing new biosorbents by immobilizing the inactive biomass of the fungus Fomitopsis pinicola by incorporating it into calcium alginate was tested. New biosorbents were prepared by liquefying inactive mushroom biomass in 1% and 2% calcium alginate, where the mushroom biomass was added in different amounts in relation to the mass of sodium alginate used in the immobilization process (10%, 20% and 30% of mushroom to the mass of alginate and in ratios of 1:1, 1:1.5 and 1:2 in relation to the mass of sodium alginate). The obtained biosorbents were dried, before and after drying, the diameter and moisture content and degree of swelling of the biosorbent microspheres were determined. Furthermore, the effectiveness of removing the synthetic dye Congo red from aqueous solutions using the obtained biosorbents was tested. The results showed that the addition of inactive mushroom biomass had an effect on increasing the diameter and reducing the water content of the obtained biosorbent microspheres compared to biosorbent microspheres without the addition of mushroom biomass. All biosorbents proved effective for removing Congo red (up to 80%) from water at the selected contact time of 6 h. Biosorbents with a reduced proportion of sodium alginate (1%) and an increased proportion of mushroom biomass also proved to be effective for the removal of Congo red, but the expected increase in efficiency compared to the sample without the addition of mushroom biomass was not achieved. The results obtained from this research point to the possibility of using waste inactive fungus Fomitopsis.pinicola for the preparation of biosorbents, but also to the need for further research that will include more detailed physico-chemical and morphological characterization of biosorbents and more extensive biosorption experiments.

Enzymes sucrose phosphorylase from Bifidobacterium adolescentis (BaSP), cellobiose phosphorylase from Cellulomonas uda (CuCBP) and cellodextrin phosphorylase from Clostridium cellulosi (CcCDP) were co-expressed in E. coli BL21 (DE3) -agp⁻ pPOLY_new cells and immobilized in polyacrylamide matrix (PAM) with the aim of repeated use of immobilized cells, i.e. three enzymes in the production of soluble cellodextrins (COS). Three repetitive batch experiments were performed at three different temperatures (35, 40 and 45 °C), differing in number of cycles (9, 6 and 5, respectively), duration of each cycle (8, 12 and 6 h), cell loading (63, 40, 40 mg CDW/g PAM-particles) and cell catalyst concentration (4, 3, 4 g CDW/L). The impact of these variables has been investigated and a new experimental setup for the industrial production of COS has been proposed; six repetitive batch cycles should be performed at 40 °C, with a duration of each cycle 8-10 h, with cell loading higher than 40 mg CDW/g PAM-particles, and concentration of the cell catalyst (higher than 4 g CDW/L). Enzimi saharoza fosforilaza iz Bifidobacterium adolescentis (BaSP), celobioza fosforilaza iz Cellulomonas uda (CuCBP) i celodekstrin fosforilaza iz Clostridium cellulosi (CcCDP) su ko-eksprimirani u stanicama E. coli BL21 (DE3) -agp- pPOLY_new. Stanice su zatim imobilizirane u poliakrilamidnom matriksu (PAM) s ciljem ponavljajućeg korištenja imobiliziranih stanica, odnosno ova tri enzima u proizvodnji topljivih celodekstrina (COS). Proizvodnja COS pomoću PAM imobiliziranih stanica tijekom ponavljajućih šaržnih eksperimenata provedena je na tri različite temperature (35, 40 i 45 °C) s različitim brojem ciklusa (9, 6 i 5), vremenom trajanja ciklusa (8, 12 i 6 h), udjelom stanica (63, 40 , 40 mg s.t./g immobilizata) i koncentracijom stanica (4, 3 , 4 g s.t./L). Utjecaj ovih parametara je ispitan i predloženi su sljedeći uvjeti za provođenje ponavljajućih šarži za industrijsku proizvodnju COS-a; šest ponavljajućih šaržnih ciklusa treba provesti na 40 °C, s trajanjem ciklusa 8-10 h, s većim udjelom stanica u poliakrilamidnom matriksu (≥ 4 g s.t./L), i koncentracijom stanica (≥ 63 s.t./g immobilizata).

Hidrogelovi predstavljaju polimerne mreže sa hidrofilnim osobinama, sposobne da apsorbuju velike količine vode, uz povećanje svoje zapremine. Geliranje se može izvesti fizičkim ili hemijskim tipovima umrežavanja polimernih lanaca, koji mogu da vode poreklo od prirodnih i sintetičkih polimera. U oblastima biomedicine, najzastupljeniji su hidrogelovi dobijeni od prirodnih polimera, kao što su polisaharidi i njihovi derivati. Cilj ovog rada je bio modifikovati različite polisaharide (alginat, pektin, karboksimetilceluloza) fenolnim jedinjenjima (tiramin, tirozin, dopamin, L-DOPA), sa ciljem dobijanja polimera sposobnih da grade hidrogelove. Uspešnost modifikacije je okarakterisana UV-Vis, FTIR i 1H NMR spektroskopskim metodama. Ispitana je njihova sposobnost geliranja, nakon čega su oni sa najpogodnijim karakteristikama primenjeni u različitim oblastima. Dopaminski derivati alginata su korišćeni za enkapsulaciju ćelijskih zidova sa enzimom lakazom, dobijenih lizom ćelija Saccharomyces cerevisiae sa prethodno eksprimiranom lakazom na površini ćelije. Dobijeni ćelijski zidovi sa lakazom su pokazali 300 puta veću enzimsku aktivnost u odnosu na nelizirane ćelije. Uspešno je urađena imobilizacija u kalcijum-dopamin-alginatnim kuglicama, koje su pokazale prednost u odnosu na kuglice sa nativnim alginatom. Ovako dobijeni biokatalizatori su korišćeni za dekolorizaciju tekstilnih boja Amido Black 10B, Reactive Black 5, Evans Blue, i Remazol Brilliant Blue. Nakon 10 ciklusa dekolorizacije od po 48 sati, efikasnost dekolorizacije za boje Evans Blue i Amido Black 10 B je iznosila 90 % i 61 %, navedenim redom. Lakaza iz Streptomyces cyaneus je takođe eksprimirana u E. coli, izolovana i prečišćena. Izolovana lakaza je imobilizovana u dopamin-pektinskim mikrokuglicama, metodom enzimske polimerizacije u emulziji. Imobilizovana lakaza je pokazala povećanu termalnu i pH stabilnost u odnosu na slobodan enzim, kao i sposobnost dekolorizacije boja Amido Black 10B, Reactive Black 5 i Evans Blue. U uzastopnim ciklusima dekolorizacije boja je ovaj biokatalizator pokazao najviše potencijala za degradaciju boja Amido Black 10B i Reactive Black 5. Sintetisani dopaminski derivati karboksimetilceluloze i alginata su korišćeni za proizvodnju nanovlakana elektrospining metodom. Dobijena nanovlakna su dodatno umrežavana i korišćena kao podloga za gajenje ćelija fibroblasta (MRC-5). Dopamin-alginatna nanovlakna su se pokazala kao dobar materijal za gajenje ćelija, što je pokazano snimanjem SEM-a i MTT esejem, iz čega bi moglo da se zaključi da su adhezija i proliferacija ćelija moguće na ovim nanovlaknima i da su ona potencijalno dobar materijal za tkivni inženjering. Hydrogels are polymer networks, which have hydrophilic properties and could absorb large amount of water, increasing their volume. They could form gels either by physical or chemical cross-linking of polymer chains and could be derived from natural and synthetic polymers. The aim of this study was to modificate diverse polysaccharides (alginate, pectin, and carboxymethylcellulose) with phenol compounds (tyramine, tyrosine, dopamine, L-DOPA), in order to obtain polymers which could form hydrogels. The success of modification was confirmed by UV-Vis, FTIR and 1H NMR spectroscopy. Their gelling ability was investigated, afterward polymers with the most suitable characteristics were applied in different areas. Dopamine alginate derivatives were used to encapsulate cell walls with the enzyme laccase, obtained by lysing cells of Saccharomyces cerevisiae, with previously expressed laccase on the cell surface. The obtained cell wall laccase has shown 300 times higher enzyme activity, compared to the whole cells. Immobilization was successfully performed in calcium-dopamine-alginate beads, which showed an advantage compared to beads with native alginate. Thus obtained biocatalyst were used for decolorization of textile dyes Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue. After ten cycles of repeated use, decolourization efficiency for dyes Evans Blue and Amido Black 10B was 90 % and 61 %, respectively. Laccase from Streptomyces cyaneus was also expressed in E. coli, isolated and purified. The isolated laccase was immobilized in dopamine-pectin microbeads, using emulsion-based enzymatic crosslinking polymerization. The immobilized laccase showed improved thermal and pH stability in comparison to the free enzyme, and ability of decolorization dyes Amido Black 10B, Reactive Black 5 and Evans Blue. In successive cycles of dyes decolorization, this biocatalyst showed the greatest potential for degradation of Amido Black 10B and Reactive Black 5. The synthesized dopamine derivatives of carboxymethylcellulose and alginate were used for the production of nanofibers by the method of electrospinning. The obtained nanofibers were additionaly crosslinked and used as a scaffold for fibroblast cells (MRC-5) cultivation. Dopamin-alginate nanofibers have been shown as a good material for cell cultivation, as shown by SEM imaging and MTT assay. It could be concluded that cell adhesion and proliferation are possible on these nanofibers and they are potentially good material for tissue engineering.

Proizvodnja piva tehnologija je koja se, kroz posljednjih nekoliko desetljeća, nije puno mijenjala. Ipak, suvremeni proces proizvodnje piva otvorio je mogućnost primjene imobiliziranih stanica u fermentaciji, a time i mogućnost korištenja novih tipova bioreaktora. Jedan od takvih primjera jest postupak ubrzane fermentacije piva imobiliziranim stanicama kvasca u bioreaktoru s unutarnjom cirkulacijom. Saccharomyces uvarum, pivski kvasac donjeg vrenja, imobiliziran je smještanjem stanica u matricu alginatnog gela u vidu čestica sfernoga oblika. Imobilizacijom velikog broja stanica ili maloga broja stanica kvasca, kojima je omogućen rast unutar alginatnih čestica, moguće je bilo dobiti visoku koncentraciju aktivnih stanica kvasca. Primjenom bioreaktora s unutarnjom cirkulacijom izvedena je serija šaržnih fermentacija ohmeljene pivske sladovine, gdje je fermentacija trajala od dvanaest do osamnaest sati, što znači da je jedan od potencijala primjene imobiliziranih stanica znatno skraćivanje vremena trajanja procesa u odnosu na tradicionalan način proizvodnje piva. U okviru ovoga rada stavlja se naglasak na postupak fermentacije piva u tradicionalnom smislu, opis metabolizma stanica kvasca, promjene u sastavu supstrata kao posljedica djelovanja stanica kvasca i njihovoj primjeni u pivarstvu. Beer production is an old technology that has not changed so much over the last few decades. However, the modern process of beer production has opened up the possibility of using immobilized cells in fermentation, and thus the possibility of using new types of bioreactors. One such example is the process of accelerated fermentation of beer by immobilized yeast cells in an internal loop gas-lift bioreactor. Saccharomyces uvarum, bottom-fermenting brewer's yeast is immobilized by placing the cells into an alginate gel matrix in the form of spherical particles. By immobilizing a large number of cells or a small number of yeast cells that have been allowed to grow within alginate particles, it is possible to obtain a high concentration of active yeast cells. Using a bioreactor with internal loop gas-lift, a series of batch fermentations of hop beer malt were performed, where the fermentation lasted from 12 h to 18 h, which means that one of the potentials of the application of immobilized cells is a significant reduction in the duration of the process compared to the traditional way of beer production. In this paper emphasis is put on the process of beer fermentation in the traditional sense, the description of yeast cell metabolism and changes in substrate composition as a consequence of their action and their important application in brewing.

Prekursor bočnog lanca statina koji je jedan od najtraženijih farmaceutika se sintetizira korištenjem kemijskih i biokatalitičkih metoda. Aldolna adicija acetaldehida i kloroacetaldehida katalizirana enzimom deoksiriboza-fosfat aldolazom (DERA) (slika 1) je metoda čija je prednost proizvodnja enantiomerno čistog produkta, ali veliki nedostatak je deaktivacija enzima DERA u prisutnosti aldehida. Imobilizacija enzima na čvrsti nosioc je jedan od načina za povećanje stabilnosti enzima. Modeliranje kinetike reakcije, te uspostavljanje matematičkog modela procesa omogućuje razvoj i intenzifikaciju enzimski katalizirane reakcije. U ovom radu je enzim DERA imobiliziran na magnetske nanočestice kovalentnim vezanjem. Za funkcionalizaciju nosioca korišten je 3-aminopropiltrietoksilan (APTES), a za aktivaciju benzokinon. Kinetička karakterizacija DERA-e je provedena u prvom i drugom stupnju reakcije. U oba slučaja kinetika je opisana dvosupstratnom Michaelis-Menteničinom jednadžbom. Ispitana je i deaktivacija enzima u prisutnosti različitih koncentracija acetaldehida i kloroacetaldehida.

U ovom radu provedena je imobilizacija enzima DERA na mezoporoznu siliku MCF funkcionaliziranu s (3– aminopropil)trimetoskisilanom u svrhu povećanja njegove stabilnosti u reakciji dvostruke aldolne adicije acetaldehida i kloroacetaldehida pri čemu nastaje prekursor u proizvodnji statina. Određivao se utjecaj procesa aktivacije nosioca na uspješnost imobilizacije korištenjem različitih aktivacijskih agenasa pri različitim koncentracijama: benzokinon (1, 5, 3 i 4, 5 mM), sukcinski anhidrid (5, 10 i 15 %) i glutaraldehid (10, 15 i 20 %). Najviša vrijednost parametara uspješnosti imobilizacije je zabilježena korištenjem 10 % sukcinskog anhidrida te je nosioc u daljnjim ispitivanjima aktiviran na taj način. Ispitan je utjecaj pH u području 6-8, te temperature u području 20-35°C na proces imobilizacije. Kao optimalni uvjeti su se pokazali pH 6 i temperatura od 30 °C. Pri tim uvjetima je provedena imobilizacija enzima te je praćena njegova aktivnost kroz pet ciklusa u reakciji sinteze bočnog lanca statina. Rezultati su pokazali kako imobilizirani enzim zadržava 43 % svoje aktivnosti nakon petog ciklusa dok je kod slobodnog enzima prisutan potpuni gubitak aktivnosti već nakon drugog ciklusa.

Bioplin nastaje procesom anaerobne pretvorbe organskih materijala uz prisustvo anaerobnih organizama. Jedno od biotehnoloških rješenja za proizvodnju bioplina su reaktori s aktivnim muljem koji koriste poljoprivrednu energetsku kulturu Miscanthus x giganteus kao alternativnu sirovinu kukuruznoj silaži. Cilj rada je bioaugmentirati reaktore za proizvodnju bioplina s ciljem povećanja njihove učinkovitosti. Bakterije koje smo uspjeli izolirati iz aktivnog mulja koristile su M. giganteus kao jedini izvor ugljika. Nakon izolacije bakterije uspješno su se imobilizirale na perlit biofilmom te tako su tvorile biočestice. Postavljanjem 9 različitih bioreaktora i njihovim praćenjem proizvodnje bioplina ukazalo se da nema značajnih razlika u proizvodnji bioplina između reaktora s biočesticama, bakterijskom suspenzijom i aktivnog mulja s dodatkom M. giganteus. Predlaže se daljnje i opširnije istraživanje s više replika te s različitim koncentracijama biočestica, da bi se vidjelo može li se povećati prinos bioplina u nekoj drugoj konfiguraciji, ili možda da se poveća stabilnost različitih bioreaktora. Biogas is produced during the process of anaerobic conversion of organic materials in the presence of anaerobic organisms. A biotechnological solution for biogas production is active sludge reactor using the agricultural energy crop Miscanthus x giganteus as an alternative raw material to corn silage. The aim of this paper is to bioaugment production reactors biogas in order to increase their efficiency. Successfully isolating the bacteria from activated sludge required using M. giganteus as the only carbon source. After isolating the bacteria, they were successfully immobilized on perlite using biofilm and subsequently formed bioparticles. Setting 9 different bioreactors and monitoring biogas production indicated that there are no significant differences in biogas production between bioparticle reactors, bacterial suspension and activated sludge with added M. giganteus. The recommendation is to undertake further and more extensive research with more replications and different concentrations of bioparticles, in order to ascertain whether biogas can be increased in another configuration, or perhaps increase the stability of different types of bioreactors.

Encimska kataliza omogoča izvedbo enantioselektivnih reakcij pri nizkih temperaturah, blagem pH in atmosferskem tlaku. Kljub tem prednostim pa je za ekonomsko upravičenost biokatalitskih procesov v industriji ključna imobilizacija encimov. Ta zagotovi manjše izgube dragih encimov, hkrati pa lahko izboljša njihovo stabilnost in odpornost v pogojih industrijskih procesov, ki zelo odstopajo od naravnega okolja encimov. Izvedba biokatalitskih procesov v mikropretočnih sistemih, ki zaradi dobre regulacije temperature, prenosa snovi in majhnega volumna omogočajo visoke konverzije ob majhnih količinah porabljenih topil ter nastalih stranskih produktov, hkrati pa nudijo možnost kontinuirnega delovanja, v zadnjem času zavzema vse več pozornosti. Prepletena nanovlakna predstavljajo prilagodljiv nosilec za imobilizacijo encimov v mikroreaktorje. Imajo veliko specifično površino z možnostjo funkcionalizacije, obenem pa zaradi velike poroznosti omogočajo skoraj nemoten transport snovi. V pričujočem delu smo izrazili in izolirali več amin transaminaz s heksahistidinskm (His6) označevalcem na različnih koncih proteina in brez njega ter primerjali njihovo imobilizacijo na prepletenih nanovlaknih z različnimi funkcionalnimi skupinami. Na nanovlaknih, na katere se je transaminaza vezala kovalentno, smo opazili veliko izgubo aktivnosti, medtem ko je koordinativna vezava preko His6 označevalca zadržala največ prvotne encimske aktivnosti. Poleg tega smo na izbranih prepletenih nanovlaknih uspešno izvedli koimobilizacijo kofaktorja encima. Enzyme catalysis allows for enantioselective reactions to be performed at low temperatures, mild pH and atmospheric pressure. Despite these advantages, the immobilization of enzymes is crucial for the economic viability of biocatalytic processes in industry. Immobilization ensures lower losses of expensive enzymes and at the same time can improve their stability under the conditions of industrial processes that deviate greatly from the natural environment of enzymes. The implementation of biocatalytic processes in microflow systems allows for high conversions with small amounts of solvents and by-products, due to good temperature regulation, fast mass transfer and low volume. At the same time microflow systems offer the possibility of continuous operation and have received much attention lately. Nanofiber mats represent a flexible carrier for the immobilization of enzymes in microreactors. They have a large specific surface with the possibility of functionalization and at the same time, due to their high porosity, they enable efficient mass transport. In this work, expression and isolation of several amine transaminases with and without hexahistidine (His6) tags at different ends of the protein was performed and compared regarding their immobilization efficiency on nanofiber mats with different functional groups. A large loss of enzyme activity was observed when transaminases were covalently bound to the nanofiber mat, while coordinate binding via His6 tag resulted in the highest retained enzyme activity. In addition, the cofactor co immobilization on a selected nanofiber mat was successful.

U disertaciji su prikazani rezultati ispitivanja primene ubrzane karbonizacije kao tehnike za unapređenje tretmana solidifikacije/stabilizacije sedimenta zagađenog teškim metalima.U prvom delu istraživanja izvršena je optimizacija metode ubrzane karbonizacije koja je korišćena za unapređenje tretmana solidifikacije/stabilizacije zagađenog sedimenta. U drugom delu istraživanja sprovedeni su tretmani solidifikacije/stabilizacije (S/S) i solidifikacije/stabilizacije sa ubrzanom karbonizacijom (S/S/K) zagađenog sedimenta kanala Begej, koji je prethodno prošao elektrokinetički tretman. Kao imobilizacioni agensi korišćeni su biopepeo od pšenične i sojine slame i biopepeo od melase. Nakon tretmana na monolitnim smešama izvršena je procena rizika primenom sekvencijalnog postupka ekstrakcije i testova izluživanja DIN 3841-4 S4 i TCLP. Istovremeno je procenjen uticaj ubrzane karbonizacije na efikasnost S/S tretmana kao i odabir efikasnijeg imobilizacionog agensa radi opširnijeg ispitivanja njegove primene u tretmanima S/S i S/S/K sedimenata Velikog bačkog kanala dodatno zagađenog metalima. Pored biopepela od pšenične i sojine slame kao imobilizacioni agens u ovom delu je korišćen i kalcijum-oksid. Testovi izluživanja i sekvencijalna ekstrakcija pokazali su da ubrzana karbonizacija pozitivno utiče na imobilizaciju kritičnih metala (Zn, Cu i Cd) u monolitnim smešama, što je u korelaciji sa povećanjem karbonatne faze (potvrđeno XRD i SEM/EDS analizom), kao i nižom stopom toksičnosti monolita u odnosu na zagađeni sediment. Na osnovu primenjenih DIN 3841-4 S4 i TCLP testova sve monolitne smeše se kategorišu kao neopasne i bezbedne za odlaganje sa aspekta izluženih Zn, Cu, Cd i Ni u skladu sa relevantnim srpskim Uredbama. Novorazvijena metoda S/S/K zagađenog sedimenta omogućava upotrebu velikih količina biopepela (upotreba otpada) i smanjenje emisije ugljen-dioksida u atmosferu njegovim dugoročnim skladištenjem i sekvestracijom u monolitnim smešama bezbednim za odlaganje., This dissertation investigates the application of accelerated carbonization as a technique for improving the solidification/stabilization treatment of sediment contaminated with heavy metals. In the first part of the research, the accelerated carbonization method is optimised for use to improve the treatment of solidification/stabilization of contaminated sediment. In the second part of the research, solidification/stabilization (S/S) and solidification/stabilization with accelerated carbonization (S/S/K) treatments are applied to contaminated sediment of the Begej canal which had previously undergone electrokinetic treatment. Wheat and soybean straw bio-ash and molasses bio-ash were used as immobilizing agents. After treatment of the monolithic mixtures, risk assessment was performed using a sequential extraction procedure and the DIN 3841-4 S4 and TCLP leaching tests. The influence of accelerated carbonization on the efficiency of S/S treatment was assessed, and the most efficient immobilizing agent was investigated, in order to provide an in depth insight into the application of the S/S and S/S/K treatments on the metals-contaminated sediments of the Veliki bački canal. In addition to bio-ash from wheat and soybean straw, calcium oxide was also used as an immobilizing agent. Leaching tests and the sequential extraction showed that accelerated carbonization has a positive effect on the immobilization of critical metals (Zn, Cu and Cd) in monolithic mixtures, which correlates with an increase in carbonate phase (confirmed by XRD and SEM/EDS analysis), as well as a lower toxicity of the monolith relative to the contaminated sediment. The results of the applied DIN 3841-4 S4 and TCLP tests show that all the monolithic mixtures are categorized by the relevant Serbian Regulations as non-hazardous and safe for disposal, based on the leached concentrations of Zn, Cu, Cd and Ni. The newly developed S/S/K method for treating polluted sediment enables the use of large amounts of bio ash (waste use) and the reduction of carbon dioxide emissions into the atmosphere by its long-term storage and sequestration in monolithic mixtures which are safe for disposal.

V zadnjih letih se je izkazalo, da imajo samosestavljivi (angl. self-assembling) hidrogeli velik potencial za uporabo v biotehnologiji, saj med drugim omogočajo zelo učinkovito imobilizacijo biokatalizatorjev, še posebno v okviru mikroreaktorjev. V delu sem predstavil osnovne koncepte mikroreaktorjev, njihove prednosti pred konvencionalnimi reaktorji ter njihovo uporabo. Poleg opisa različnih načinov imobilizacij biokatalizatorjev v mikroreaktorje, kot so imobilizacija na površino mikroreaktorja, zamreženje v gel in imobilizacija z magnetnimi delci, sem natančneje predstavil samosestavljive hidrogele. Delo obravnava pregled literature s področja različnih načinov priprave tovrstnih hidrogelov, kot tudi njihove uporabe, predvsem pri kontinuirni sintezi snovi z encimskimi mikroreaktorji. In recent years, self-assembling hydrogels have been shown to have great potential for use in biotechnology, as they enable very efficient immobilization of biocatalysts, especially when used within microreactors. In this work, the basic concepts of microreactors, their advantages over conventional reactors and their potential applications are presented. Different techniques for biocatalyst immobilization in microreactors are described, such as surface immobilization, crosslinking and immobilization on magnetic particles, while self-assembling hydrogels are presented in more detail. The work includes an overview of the methods for the preparation of self-assembling hydrogels and their use in continuous synthesis with enzymatic microreactors.

Fraktura ili prijelom je prekid kontinuiteta kosti, a koja najčešće nastaje kod intenzivnog i kratkog vanjskog opterećenja. Lom kosti nastaje djelovanjem vanjske sile koja je veća od njihove čvrstoće i obično nastaje zbog jakih naprezanja. Prijelomi se dijele na otvorene i zatvorene, a na samom području prijeloma nastaje oteklina, podljev krvi, crvenilo ili deformitet ekstremiteta. Najčešći prijelomi koji nastaju su prijelomi ručnog zgloba, stopala i šake, dok prijelomi dugih kostiju poput kostiju ruku i nogu nastaju djelovanjem jače sile, npr. kod prometnih nesreća. Prvi korak kod zbrinjavanja prijeloma je procijeniti težinu i vrstu istog, dovesti ekstremitet do stanja prihvatljivog za imobilizaciju, provesti imobilizaciju, te spremiti pacijenta za transport u bolnicu na daljnje liječenje. Vrlo je važno poznavati simptome i znakove prijeloma kako bi se što brže interveniralo, jer što bržim pružanjem same prve pomoći, veće su šanse za kvalitetniju zdravstvenu skrb pacijenta i ozlijeđenog dijela tijela. Veliku ulogu u liječenju imaju medicinske sestre/tehničari „na terenu“ koji najprije zbrinjavaju pacijenta na mjestu nesreće i transportiraju ga u bolnicu, pa tako i oni/one „u bolnici“ koji prate pacijenta od prijema pa sve do njegove konačne rehabilitacije. A fracture is an interruption of bone continuity, which most often occurs with intense and short external loads. Bone fractures are caused by an external force that is greater than their strength and usually occur due to strong stresses. Fractures are divided into open and closed, and in the area of the fracture there is swelling, bruising, redness or deformity of the extremities. The most common fractures that occur are fractures of the wrist, foot and hand, while fractures of long bones such as the bones of the arms and legs are caused by a stronger force, eg in traffic accidents. The first step in caring for a fracture is to assess the severity and type of the fracture, bring the limb to a condition acceptable for immobilization, perform immobilization, and prepare the patient for transport to the hospital for further treatment. It is very important to know the symptoms and signs of a fracture in order to intervene as quickly as possible, because the faster the first aid is provided, the better the chances for better health care for the patient and the injured part of the body. Nurses / technicians "in the field" have a big role in the treatment, who first take care of the patient at the scene of the accident and transport him to the hospital, as well as those "in the hospital" who follow the patient from admission to his final rehabilitation.

Cellulases are enzymes which catalyse cellulose hydrolysis and are widely used in various industry branches. Lately, their application in treatment of different agroindustrial waste materials which could serve for fuel production is being extensively explored. In order to increase their stability and cost-effectiveness of their usage, application of their immobilized forms are preferred over free enzymes. Hereby, we tested eight different LifetechTM immobilization supports differing in polarity, porosity and functional groups as carriers for Asspergillus Niger cellulase immobilization. Most promising carrier was methacrylate based, with primary amino groups, C6 "space arm" and pores with diameter of 60-120 nm - LifetechTM ECR8409F. For this support, most important immobilization parameters were investigated and after 3 h at pH 6 with initial protein concentration of 23.3 mg/g support immobilized cellulase with 406 IU/g (with carboxymethyl cellulose as a substrate) was obtained. This preparation was successfully applied in the hydrolysis of lignocellulosic fraction of sunflower seed meal, which is widely available byproduct of sunflower seed meal fractionation for protein-rich fractions production. Initial reaction rates and yields of reducing sugars were unchanged comparing to free enzyme, indicating that there were no significant diffusion limitations for substrate to approach active sites of A. Niger cellulase molecules immobilized onto LifetechTM ECR8409F support. Celulaze su enzimi koji katalizuju hidrolizu celuloze i u širokoj su primeni u brojnim granama industrije. U poslednje vreme, mogućnost njihove primene u tretiranju različitih opadnih agroindustrijskih sirovina u cilju dobijanja biogoriva intenzivno se istražuje. Da bi se povećale stabilnost i ekonomičnost njihove primene, potrebno je umesto slobodnog enzima koristiti imobilisane forme. U ovom istraživanju osam LifetechTM nosača različitih polarnosti, poroznosti i funkcionalnih grupa. testirano je za imobilizaciju celulaze producenta Aspergillus Niger. Najpogodniji nosač bio je na bazi metakrilata, sa primarnim amino grupama, C6 "dugom nožicom" i porama prečnika 60-120 nm - LifetechTM ECR8409F. Za ovaj nosač određeni su najznačajniji uslovi imobilizacije i nakon 3 sata na pH 6, pri početnoj koncentraciji proteina od 23,3 mg/g nosača dobijena je imobilisana celulaza aktivnosti 406 IU/g (sa karboksi metil celulozom kao supstratom). Ovaj preparat uspešno je primenjen u reakciji hidrolize lignocelulozne frakcije suncokretove sačme, koja prestavlja nusproizvod frakcionisanja suncokretove sačme pri dobijanju frakcija bogatih proteinima. Početne brzine reakcije i prinosi redukujućih šećera bili su nepromenjeni u odnosu na slobodan enzim, ukazujući da nije bilo značajnog uticaja difuzionih limitacija pri prilasku supstrata aktivnim centrima molekula A. Niger celulaze imobilisane na LifetechTM ECR8409F nosač.

Prolonge d periods of complete physical inactivity or bed rest trigger various alterations in the functional and metabolic levels of the human body. However, bed rest-related adaptations of the central nervous system are less known and thoroughly studied. The aim of this study was to investigate brain electrophysiological changes using event-related potentials (ERPs) after 14 days of bed rest and 12 consecutive sessions of computerized cognitive training (CCT). Sixteen older (Mage= 60 years) healthy volunteers were randomly divided into a CCT treatment group and an active control group. All participants performed ERP measurements based on the foveal visual presentation of a circle on a black background before and after bed rest. After 14 days of bed rest, participants in the control group showed increased peak P1 amplitude (p = .012), decreased P1 latency (p = .024), and increased P2 amplitude (p = .036), while the CCT group also showed decreased P1 latency (p = .023) and decreased P2 latency (p = .049). Our results suggest that, even from a central adaptation perspective, prolonged periods of physical inactivity or bed rest trigger additional neural recruitment and should therefore be minimized, and that CCT may serve as a tool to mitigate this. Future research should focus on other aspects of central nervous system adaptation following periods of immobilization/hospitalization to improve our knowledge of infl uence of physical inactivity and its eff ects on cortical activity and to develop appropriate countermeasures to mitigate functional dysregulation. Dolgotrajna obdobja popolne gibalne neaktivnosti ali horizontalnega ležanja sprožijo v človeškem telesu različne spremembe na funkcionalni in metabolni ravni. Prilagoditve centralnega živčnega sistema, povezane s horizontalnim ležanjem, so manj poznane in še ne dovolj preučene. Namen te raziskave je bil oceniti možganske elektrofi ziološke spremembe z uporabo metode z dogodkom povezanih potencialov (ERP) po 14-dnevnem horizontalnem ležanju in 12 zaporednih vadbah računalniškega kognitivnega treninga (RKT). Šestnajst starejših (Mstarost= 60 let) zdravih prostovoljcev je bilo naključno razdeljenih v intervencijo RKT in aktivno kontrolno skupino. Vsi udeleženci so izvajali meritve ERP pred in po horizontalnem ležanju na podlagi fovealne vidne predstavitve kroga na črni podlagi. Po 14-dnevnem horizontalnem ležanju je analiza ERP pokazala povečano amplitudo P1 (p = ,012), zmanjšano latenco P1 (p = ,024) in povečano amplitudo P2 (p = ,036) pri kontrolni skupini, medtem ko sta se v skupini RKT latenci P1 (p = ,023) in P2 skrajšali (p = ,049). Naši rezultati kažejo, da daljša obdobja gibalne neaktivnosti ali horizontalnega ležanja sprožijo, tudi z vidika centralne prilagoditve, dodatno rekrutacijo nevronov, zato je treba taka obdobja zmanjšati na najmanjšo možno mero. Ugotovljeno je bilo tudi, da lahko RKT služi kot orodje za ublažitev upada. Prihodnje raziskave bi se morale osredotočiti še na druge vidike prilagajanja centralnega živčnega sistema po obdobjih imobilizacije/hospitalizacije, da bi izboljšali razumevanje posledic gibalne neaktivnosti in njenih učinkov na kortikalno aktivnost ter razvili ustrezne protiukrepe za blaženje funkcionalne disregulacije.

Polietilenimin (PEI) sintetski je kationski polimer čija funkcionalnost i reaktivnost potječu od činjenice da se radi o polimeru koji sadrži primarne, sekundarne i tercijarne skupine amina. Zbog ovakve građe, PEI ima veliku gustoću naboja što omogućuje postizanje jednakog učinka imobilizacije enzima uz manju količinu polimera, a da pri tome ne dođe do aglomeracije enzima. Ovaj je polimer fleksibilan i jednostavan za uporabu, pristupačne je cijene te omogućuje dodatnu uštedu zbog mogućnosti ponovne uporabe enzima nakon imobilizacije. PEI koristi se u brojne svrhe, primjerice kao ljepilo za imobilizaciju enzima i ko-imobilizaciju višeslojnih enzima, kao sredstvo za imobilizaciju enkapsuliranjem te za pripremanje imobilizacijskih podloga. Svojstva polietilenimina, načini njegova dobivanja i primjeri uporabe u industriji bit će detaljnije prikazani u nastavku ovoga završnog rada. Polyethylenimine (PEI) is a synthetic cationic polymer whose functionality and reactivity arise from the fact that it contains primary, secondary and tertiary amino groups. Due to such structure, PEI has a high charge density which enables an effective enzyme immobilization with a smaller amount of polymer, while disabling enzyme agglomeration. This polymer is flexible, easy to use and it is affordable which allows additional savings due to the possibility of immobilized enzyme reuse. PEI is used for many purposes, e.g. as an immobilization glue, for co-immobilization of enzyme multilayers, for enzyme encapsulation and for preparation of immobilization supports. PEI properties, methods of its production, as well as some of the examples of its use in industry will be presented in this undergraduate thesis.

Lipaze, triacilglicerol-acilhidrolaze, su zbog svoje svestranosti djelovanja našle primjenu u prehrambenoj industriji, industriji kože i tekstila, ulja i masti, papira te u kozmetičkoj i farmaceutskoj industriji. Iako je primjena lipaza u odnosu na kemijske katalizatore poželjnija, zbog visoke specifičnosti obzirom na supstrat i tip reakcije, ekološku prihvatljivost i energetsku nezahtjevnost, visoka cijena lipaza kao i vrlo česta nemogućnost ponovne uporabe slobodnih lipaza predstavlja ključnu prepreku za širu primjenu u industrijskoj proizvodnji. Navedeno se može nadići primjenom imobiliziranih lipaza, koje se po završenom proizvodnom procesu mogu lako izdvojiti iz reakcijske smjese te ponovo uporabiti u sljedećem proizvodnom procesu ili u kontinuiranim procesima proizvodnje. Za imobilizaciju enzima danas se koriste različite tehnike koje se mogu svrstati u tri osnovne skupine: i) imobilizaciju adsorpcijom, ii) imobilizaciju direktnim ili indirektnim kovalentnim vezanjem, te iii) imobilizaciju zarobljavanjem enzima. Obzirom da većina komercijalno dostupnih nosača ima relativno visoku cijenu, postoji potreba za pronalaskom jeftinijih. Među brojnim otpadom poljoprivredno- prehrambene industrije kao potencijalni kandidati za proizvodnju nosača izdvajaju se ljuska jaja, talog kave i ljuska crvenog luka koji još nisu pronašli odgovarajuće mjesto u održivoj proizvodnji, nego se uglavnom odlažu na polja i odlagališta te na takav način dodatno opterećuju okoliš. Primjenom inovativnih tehnika transformacije otpada mogli bi se proizvesti nosači na bazi celuloze i/ili hemiceluloze (talog kave i ljuska crvenog luka) ili nosači na bazi kolagenske mreže (membrana ljuske jaja). Za očekivati je da će se u sljedećih nekoliko desetljeća morati pronaći nove i/ili poboljšati postojeće tehnologije koje će slijediti trend održive proizvodnje obzirom na porast interesa za proizvodima koji su dobiveni na zdrav i održiv način.

Research background. Barberry juice is a rich source of bioactive compounds and shows different health properties such as antioxidant and anticancer activities. Clarification, as the removal process of suspended material, is an important step in the production of fruit juice due to its significant effect on the appearance, flavour and commercialisation of juice. Pectinase is the most important enzyme applied in juice clarification that breaks down the pectin polymer structure and reduces the undesirable turbidity. Pectinase immobilisation is a way to overcome free enzyme drawbacks such as instability, high cost, the difficulty of recovery and recyclability. Also, continuous clarification process which is highly preferred in fruit juice industry is not possible without enzyme immobilisation. Experimental approach. Pectinase enzymes were immobilised on the functionalised glass beads (glass bead with (3-aminopropyl)triethoxysilane) by glutaraldehyde, polyaldehyde derivatives of pullulan and kefiran and the barberry juice was clarified in the batch and continuous processes in a packed bed reactor (PBR). Also, the effect of clarification on the physicochemical and antioxidant properties of the barberry juice samples was evaluated. Results and conclusions. The optimum conditions for clarification in the PBR were: flow rate 0.5 mL/min, temperature 50 °C and treatment time 63 min. Clarification led to a decrease in turbidity, pH, total soluble solid content, viscosity, total phenolic content and antioxidant activity of the juice samples. Also, this process increased the clarity, acidity, reducing sugar concentration and the lightness parameter of the barberry juice. The greatest effect of clarification on the studied properties of barberry juice was related to the pectinase immobilised by the polyaldehyde of kefiran in the continuous process and both new cross-linkers (polyaldehyde derivatives of pullulan and kefiran) immobilised the enzyme better than the common cross-linker (glutaraldehyde). Novelty and scientific contribution. For the first time, barberry juice was clarified with pectinase immobilised by polyaldehyde derivatives of pullulan and kefiran and the obtained results showed that the pectinase immobilisation by these new cross-linkers was much more efficient than by the glutaraldehyde as a common cross-linker. These findings can be of use for an industrialised production of fruit juices., Pozadina istraživanja. Sok od obične žutike bogat je bioaktivnim spojevima te ima razna ljekovita svojstva, poput antioksidacijskih i antikancerogenih. Bistrenje je postupak uklanjanja materijala iz suspenzije i važan je korak u proizvodnji sokova jer bitno utječe na izgled, okus i ekonomsku isplativost soka. Pektinaza je najvažniji enzim koji se koristi za bistrenje sokova, a razgrađuje polimernu strukturu pektina i time smanjuje zamućenost soka. Imobilizacijom pektinaze uklanjaju se nedostaci slobodnog enzima, kao što su nestabilnost, visoka cijena, malo iskorištenje i slaba ponovna iskoristivost. Osim toga, kontinuirani postupak bistrenja, koji se najviše koristi u proizvodnji sokova, nije moguće provesti bez imobilizacije enzima. Eksperimentalni pristup. Pektinaza je imobilizirana pomoću glutaraldehida te polialdehidnih derivata pululana i kefirana na staklenim kuglicama prevučenim (3-aminopropil)trietoksisilanom, a sok od obične žutike bistren je šaržnim ili kontinuiranim postupkom u reaktoru s nasutim slojem nosača. Osim toga, ispitana su fizikalno-kemijska i antioksidacijska svojstva soka. Rezultati i zaključci. Optimalni uvjeti za bistrenje soka u reaktoru s nasutim slojem nosača bili su: protok 0,5 mL/min, temperatura 50 °C i trajanje postupka 63 min. Postupkom bistrenja smanjila se zamućenost soka, snizila njegova pH-vrijednost i smanjili ukupan udjel topljive tvari, viskoznost, ukupan udjel fenola te antioksidacijska aktivnost. Osim toga, povećali su se bistroća, kiselost, koncentracija reducirajućih šećera te parametri boje soka. Svojstva soka najviše su se poboljšala bistrenjem pomoću pektinaze imobilizirane polialdehidom kefirana u kontinuiranom postupku. Oba derivata polialdehida, pululan i kefiran, imobilizirali su enzim bolje od uobičajeno korištenog glutaraldehida. Novina i znanstveni doprinos. U ovom je radu po prvi put proveden postupak bistrenja soka od obične žutike pomoću pektinaze imobilizirane derivatima polialdehida pululana i kefirana. Dobiveni rezultati pokazuju da je ovako imobilizirana pektinaza bitno učinkovitija od one imobilizirane pomoću glutaraldehida, te se može upotrijebiti u industrijskoj proizvodnji voćnih sokova.

Kemijski senzori su uređaji koji kvalitativno ili kvantitativno određuju analite pretvarajući kemijsku informaciju o koncentraciji komponente uzorka u analitički koristan signal. Kod fluorescencijskih senzora mjeri se radijativni raspad, tj. emisija fluorofora. Rad senzora temelji se na selektivnom gašenju ili pojačanju intenziteta fluorescencije u dodiru s analitom (On-Off efekt). 4-amino-1,8-naftalimidi i njihovi derivati poznata su fluorescentna bojila. Postali su raznolika platforma za izradu senzora zahvaljujući svojima prednostima, poput optičkih svojstava, posebice snažna apsorbancija i emisija u vidljivom dijelu spektra, širok Stokesov pomak, visoka fotostabilnost i dr. Nadalje, ponašaju se kao tipični fluorofori s unutarmolekulskim prijenosom naboja (ICT), što omogućava osjetljivost na promjene u mikrookolišu. Posljednje, no jednako važno, je relativno jednostavna struktura, za čije dobivanje postoje dobro poznate sinteze. Svojstvo 1,8-naftalimidnih derivata je fotoinducirani prijenos elektrona (PET) s elektron donora na karbonilnu skupinu fluoroforske jedinice. PET smanjuje stopu radijativnog raspada. Ovisno o strukturi derivata, može doći do pojačanja ili smanjenja fluorescencije. U radu je proučen mehanizam djelovanja fluorescencijskih PET senzora temeljenih na skupini derivata 1,8-naftalimida, pri čemu utjecaj na emisiju mogu imati položaj supstituenta u strukturi, udaljenost elektron donora i akceptora, sklonost supstituenta za specifičnim interakcijama te geometrija liganada. Posebno su proučena optička svojstva u otopinama različitih kationa i pH, u svrhu izrade senzora. Navedene su metode imobilizacije na pamučnu, polimernu, staklenu podlogu, na molekulska sita i celulozne nanočestice te su uviđene prednosti kovalentne imobilizacije. Zahvaljujući pokazanim obećavajućim karakteristikama, fluorescencijski senzori temeljeni na 1,8-naftalimidima mogli bi se koristiti u realnim uzorcima vode različitog podrijetla. Chemical sensors are devices which can detect or measure analytes by converting chemical information about the concentraction of a component in a sample into an analitically useful signal. Fluorescent sensors measure radiative decay, that is emission of a fluorophore. The main characteristic of those sensors is selective quenching or increasing of the fluorescence intensity when in contact with an analyte (On-Off effect). 4-amino-1,8-naphthalimides and their derivatives are commonly used fluorescent dyes. They became versatile platforms for sensor design due to their advantageous optical properties, such as strong absorption and emission in the visible region, large Stokes shift, high photostability etc. Secondly, they are typical fluorophores with intermolecular charge transfer, which enables sensitivity to changes in the microenviroment. Lastly, but equally important, they have relatively simple structure, for which facile synthesis have been established. Property of 1,8-naphtalimide derivatives is photoinduced electron transfer (PET), which occurs from electron donor to carbonyl groups located on a fluorophore. PET process decreases the rate of the radiative decay. Depending on the structure of the derivative, either increase or quenching of fluorescence can occur. In this paper the mechanism of fluorescent PET sensors containing the derivatives od 1,8-naphthalimides is studied, where different aspects can influence its properties, such as position of the supstituent, distance of electron donor and acceptor, specific interactions and the ligand geometry. The great emphasis is on optical properties in solutions of different metal cations and pH, making the base of sensoric design. In addition, immobilisation methods to different substrates are listed; cotton fabric, polymers, glass, molecular sieves and cellulose nanoparticles, where covalent bonding has great advantages. Due to the promising characteristics shown, 1,8-naphthalimide-based fluorescence sensors could be used in actual water samples of different origins.

U ovom radu provedena je kovalentna imobilizacija biokatalizatora DERA na MCF mezoporoznu siliku funkcionaliziranu APTES-om, te aktiviranu benzokinonom u svrhu povećanja njegove stabilnosti. Mezoporozna silika sintetizirana je sol – gel metodom. Aktivnost biokatalizatora ispitana je u reakciji dvostruke aldolne adicije acetaldehida i kloroacetaldehida koja kao produkt daje laktol, važan prekursor u sintezi statina. Ispitao se utjecaj procesa aktivacije nosioca na uspješnost imobilizacije biokatalizatora korištenjem različitih koncentracija benzokinona. Rezultati su pokazali da se pri koncentraciji benzokinona od 3 mM postižu optimalne vrijednosti parametara uspješnosti imobilizacije. Također je ispitan utjecaj temperature i vremena imobilizacije na način da su ispitivanja provedena pri temperaturama od 15 - 40 °C te pri vremenima imobilizacije od 2 - 4 h. Temperatura od 25 °C se pokazala kao optimalna, a također je zaključeno kako vrijeme trajanja imobilizacije nema značajnog utjecaja na uspješnost samog procesa. In this work, the covalent immobilisation of the biocatalyst DERA in order to increase its stability was done. As a carrier MCF mesoporous silica functionalised by APTES and activated by benzoquinone was used. Mesoporous silica was synthesised by the sol–gel method. Biocatalyst activity was examined in the reaction of double aldol addition of acetaldehyde and chloroacetaldehyde, which results in lactol, an important precursor in the statin synthesis. The influence of different concentrations of benzoquinone in the activation process on the success of biocatalyst immobilization was examined. Results have shown that benzoquinone concentration of 3 mM was optimal. Also, the impact of temperature in the range of 15 - 40 °C and the immobilisation time (2 - 4 h) were investigated. Temperature of 25 °C proved to be optimal, and it was also concluded that the duration of immobilisation process had no significant impact on the success of the process.

U ovom radu provedena je imobilizacija enzima DERA na MCF mezoporoznu siliku u puferima koncetracija 0,05, 0,1 i 0,2 M pri pH vrijednostima 6, 6,5, 7, 7,5 i 8. MCF mezoporozna silika sintetizirana je sol-gel procesom. Aktivnost enzima ispitana je u reakciji aldolne adicije acetaldehida i kloroacetaldehida. Ovom reakcijom nastaje laktol, prekursor za sintezu bočnog lanca statina. Usporedbom parametara uspješnosti za navedene pufere odabrani su optimalni uvjeti za imobilizaciju enzima koji su odgovarali puferu koncentracije 0,05 M i pH vrijednosti 6,5. Imobilizacija enzima DERA potom je ispitana za nosioc većih pora u odabranom puferu. Usporedbom parametara uspješnosti oba nosioca, za nosioc većih pora dobivene su više vrijednosti efikasnosti i preostale aktivnosti. In this work enzyme DERA was immobilized on MCF mesoporous silica in various immobilization buffers. Buffers with different ionic strenght, i.e. concentration (0.05, 0.1 and 0.2 M) and pH (6, 6.5, 7, 7.5 and 8) were used. MCF mesoporous silica was synthesized by sol-gel process. Enzyme activity was measured in the reaction of aldol addition of acetaldehyde and chloroacetaldehyde. The product of this reaction is lactol, the precursor in synthesis of a statin side chain. Based on the measure of success of immobilization process, optimal immobilization buffer was the one with concentration of 0.5 M and pH 6.5. Further, the enzyme DERA was immobilized on the MCF carrier with larger pores in the optimal buffer. Higher values of immobilization efficiency and remaining activity were obtained for the carrier with larger pores.

Papirnati mikrofluidički senzori predstavljaju inovativnu tehnologiju za manipulaciju malih volumena uzoraka i detekciju razičitih vrsta analta. Cilj je takve tehnologije pružiti jeftine, bio-razgradive, lako prenosive uređaje, neovisne o skupoj instrumentaciji, a kojma će moći rukovati i needucirano osoblje. Cilj je ovog rada staviti papirnate mikrofluidičke senzore u širi kontekst mikrofluidičkih uređaja, dati pregled prednosti i mana takve tehnologije, opisati najčešće korištene metode izrade papirnatih mikrofluidičkih uređaja kao i metode detekcije analita. Velik je dio rada posvećen bitnoj skupini takvih senzora, papirnatim mikrofluidičkim biosenzorima te njihovoj podskupini, papirnatim mikrofluidičkim enzimskim biosenzorima. Rad također obrađuje područja primjene te nove tehnologije i zapreke koje bi ona trebala prevladati kako bi iz laboratorija prešla u komercijalnu upotrebu. Paper based microfluidic sensors represent an innovative technology for handling small volumes of samples and analysis of different types of analytes. The goal of this technology is to provide devices that are inexpensive, biodegradable, portable, independent of expensive equipment and that can be handled by untrained personnel. The aim of this thesis is to place paper microfluidic sensors in the wider context of microfluidic devices, discuss their advantages and disadvantages, describe the methods for their production as well as the methods for analyte detection. A considerable part of the thesis is dedicated to an important group of such sensors, paper microfluidic biosensors and their subgroup, paper microfluidic enzyme biosensors. The thesis also focuses on highlighting new technologies in the field of paper microfluidics as well as on the obstacles this technology needs to surpass in order to step out of the laboratory and becomes widely used.