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Uporaba mikroreaktorjev se je v zadnjih letih močno povečala zaradi številnih prednosti, povečala pa se je tudi uporaba bioloških katalizatorjev kot bolj trajnostni način proizvodnje. Za industrijsko rabo encimov pa je ključnega pomena uspešna imobilizacija. V tem delu smo v mikropretočnem sistemu pripravili zamrežene encimske agregate na osnovi lakaze. Pri tem smo poskušali ohraniti čim večjo aktivnost encima in pridobiti delce primerne velikosti z visoko stopnjo uniformnosti. Med seboj smo primerjali različne pogoje priprave encimskih agregatov ter uspeli zadržati do 66,8 % prvotne aktivnosti encima. Pripravljene zamrežene encimske agregate smo nato imobilizirali v membranskem mikroreaktorju. V slednjem smo poskusili izvesti biotransformacijo tirozol acetata, ki smo ga v mikroreaktor uvajali v organski fazi ob prisotnosti kisika v plinski fazi. The use of microreactors has greatly increased in recent years due to many advantages and the use of biological catalysts has also increased as more sustainable method of production. Successful immobilization is crucial for the industrial use of enzymes. In this work, crosslinked enzyme aggregates based on laccase were prepared in a microflow system. In doing so, we tried to preserve as much enzyme activity as possible and obtain particles of suitable size with high degree of uniformity. We compared different conditions for the preparation of enzyme aggregates and managed to retain up to 66.8 % of the original enzyme activity. The prepared crosslinked enzyme aggregates were then immobilized in a membrane microreactor. In the latter, we tried to carry out the biotransformation of tyrosol acetate, which was introduced into the organic phase in the presence of oxygen in the gas phase.

Namen našega dela je bil pripraviti zamrežene encimske skupke (CLEA) in magnetne zamrežene encimske skupke (mCLEA) iz encima β-galaktozidaze. Doseči smo želeli čim višjo preostalo aktivnost encima in čim višjo učinkovitost imobilizacije. Zamreženje smo preizkušali v štirih obarjalnih reagentih (acetonu, etanolu, 1-propanolu in 2-propanolu), dodajali smo ogrodne proteine in mrežne povezovalce v različnih kombinacijah in koncentracijah. Eksperimentalno delo je obsegalo pripravo vzorcev (obarjanje in zamreženje), aktivnostni test in Bradfordovo metodo za določanje koncentracije proteinov. Proces smo optimirali pri konstantni koncentraciji encima (50 mg/ml) in konstantnem dodatku glutaraldehida (GA). Ugotovili smo, da je optimalna količina dodanega mrežnega povezovalca GA 1.5% (v/v) in da mrežni povezovalec pentaetilenheksamin (PEHA) negativno vpliva na zamreženje, razen če ga dodajamo v kombinaciji z ogrodnimi proteini. V tem primeru se preostala aktivnost zviša, medtem ko učinkovitost imobilizacije močno upade. The purpose of our work was to create cross-linked enzyme aggregates (CLEA) and magnetic cross-linked enzyme aggregates (mCLEA) off enzyme β-galactosidase. The aim of our work was to achive the highest possible residual activity and maximize the efficiency of immobilization. The cross-linking was tested in four most optimal precipitation reagents (acetone, ethanol, 1-propanol, 2-propanol) while stability proteins and cross-linkers in various combinations and concentrations were added to the synthesis reaction. The experimental work included preparation of samples (precipitation and cross-linking), activity test and the Bradford protein assay for determination of proteins. The process was optimized at a constant concentration enzyme (50 mg/ml), and a constant addition of glutaraldehyde (GA). It was concluded that the optimal quantity of added cross-linker is 1.5% (v/v) and that the cross-linker pentaetilenheksamin (PEHA) negatively affects the cross-linking, unless it is added in combination with stability proteins. In this case, the residual activity is increased, while the efficacy of immobilization drops significantly.

Doktorska disertacija je razdeljena na dva sklopa, v katerih smo razširili dosedanje raziskave na področju priprave magnetnih nanodelcev, njihove funkcionalizacije in imobilizacije encimov na tako pripravljene mikro- in nanonosilce. Osrednji cilj disertacije je priprava magnetnih nanodelcev z metodo obarjanja ali koprecipitacije železovih (II) in železovih (III) ionov v alkalnem mediju, funkcionaliziranih s hitozanom po treh različnih metodah metodi mikroemulzije, suspenzijske zamreževalne tehnike in kovalentne vezave. Podrobna karakterizacija tako pripravljenih nosilcev je bila izvedena s pomočjo presevne elektronske mikroskopije, vrstične elektronske mikroskopije, rentgenske praškovne difrakcije, z dinamičnim sipanjem laserske svetlobe, laserskim granulometrom in merjenjem specifične magnetizacije. Tako smo opazovali morfologijo in lastnosti magnetnih nosilcev. Površinsko funkcionalizacijo s hitozanom za doseganje višje funkcionalnosti smo potrdili s pomočjo energijskodisperzijske spektroskopije, termične analize, potenciometrične titracije in s Fourierevo transformacijo. Z mikrobiološkim testiranjem je bil določen vpliv magnetnih mikro- in nanonosilcev na rast mikrobnih kultur. Nadalje smo magnetne mikro- in nanodelce, prevlečene s plastjo hitozana, uporabili kot nosilce za imobilizacijo holesterol oksidaze (EC 1.1.3.6). S spreminjanjem različnih pogojev, kot so koncentracija encima, koncentracija in vrsta mrežnega povezovalca glutaraldehida ali pentaetilen heksamina ter hitrost stresanja, smo ugotovili, kako se spreminja učinkovitost imobilizacije encima ter kolikšen delež aktivnosti se pri tem ohrani v primerjavi s prostim encimom. Aktivnost imobilizirane holesterol oksidaze smo določili z reakcijo oksidacije holesterola. Optimalni reakcijski pogoji imobilizacije holesterol oksidaze na magnetne mikro- in nanonosilce so bili doseženi pri koncentraciji encima 1 mg/mL ob uporabi 0,02 M pentaetilenheksamina kot mrežnega povezovalca. Aktivnost imobilizirane holesterol oksidaze pri optimalnih reakcijskih pogojih je bila 79,03 %. Prav tako je bila stabilnost imobilizirane holesterol oksidaze pri ponovni uporabi dobra. V drugem sklopu je opisana priprava zamreženih encimskih skupkov iz encima celulaze (EC 3.2.1.4). Postopek priprave zamreženih encimskih skupkov vključuje dva pomembna koraka, in sicer obarjanje encima z dodatkom obarjalnega reagenta in zamreženje encimskih molekul z glutaraldehidom. Optimalni reakcijski pogoji sinteze zamreženih encimskih skupkov so bili doseženi pri koncentraciji glutaraldehida 0,0625 % (v/v) in uporabi etanola kot obarjalnega reagenta. Aktivnost imobilizirane celulaze v obliki zamreženih encimskih skupkov smo določili z reakcijo hidrolize celuloze. Aktivnost imobilizirane celulaze v obliki zamreženih encimskih skupkov pri optimalnih reakcijskih pogojih je bila 93,95 %. Prav tako je bila stabilnost imobilizirane celulaze pri ponovni uporabi dobra. The PhD thesis is divided in two parts, in which we enhanced existing research in the filed of preparation of magnetic nanoparticles and their functionalization and enzyme immobilization on micro- and nanocarriers. The main objective of the thesis is the preparation of magnetic nanoparticles by precipitation method of iron (II) and iron (III) ions in an alkaline medium and the functionalization with chitosan by three different methods microemulsion, suspension cross-linking technique and covalent binding. Detailed characterization of these carriers was performed using transmission electron microscopy, scanning electron microscopy, X-ray powder diffraction, with dynamic laser light scattering, laser granulometry and the measurement of specific magnetization. Thus, we observe the morphology and the characteristics of magnetic carriers. Surface functionalization of the chitosan in order to achieve higher functionality was confirmed by energy dispersive spectroscopy, thermal analysis, potentiometric titration and Fourier transformation. The microbiological testing determined the influence of magnetic micro- and nanocarriers on the growth of microbial cultures. Furthermore, the magnetic nanoparticles coated with chitosan were used as a carriers for the immobilization of cholesterol oxidase (EC 1.1.3.6). By changing various conditions such as the enzyme concentration, concentration and type of cross-linker glutaraldehyde or pentaetylenehexamine and shaking rate, we determined an immobilization efficiency and retained activity of immobilized enzyme compared to free enzyme. The activity of the immobilized cholesterol oxidase was determined by reaction of the cholesterol oxidation. The optimal reaction conditions of cholesterol oxidase immobilization to the magnetic micro- and nanocarriers were obtained using 1mg/mL of the enzyme concentration and 0,02 M pentaetilenhexamine, as a cross-linker. Activity of the immobilized cholesterol oxidase under optimal conditions was 79,03 %. Also, the stability of immobilized cholesterol oxidase revealed good reusability. The second part describes the preparation of cross-linked enzyme aggregates of cellulase (EC 3.2.1.4). The preparation process of cross-linked enzyme aggregates includes two important steps, such as the precipitation of the enzyme by the addition of the precipitation reagent and the cross-linking of enzyme molecules with glutaraldehyde. The optimum reaction conditions of cross-linked enzyme aggregates of cellulase have been achieved at a 0,0625 % concentration of glutaraldehyde (v/v) and using ethanol as a precipitation reagent. The activity of immoblized cellulase as cross-linked enzyme aggregates was determined by reaction of hydrolysis of cellulose. Activity of the immobilized cellulase as cross-linked enzyme aggregates under optimal conditions was 93,95 %. Also, the stability of immobilized cellulase revealed good reusability.

Namen diplomske naloge je sinteza maghemitnih nanodelcev, prevlečenih z aminosilanom, ter imobilizacija encima lipaze na pripravljen nosilec. Maghemitne nanodelce smo pripravili z obarjalno reakcijo ali koprecipitacijo železovih II in železovih III ionov s 25 % amonijakom in jih prevlekli s funkcionalno plastjo silike, nato pa še s plastjo aminosilana. Optimirali smo pogoje za dosego najvišje učinkovitosti imobilizacije ter preostale aktivnosti imobiliziranega encima, kot so: koncentracija encima, vrsta mrežnega povezovalca, čas imobilizacije ter koncentracija mrežnega povezovalca glutaraldehida. Proučevali smo stabilnost imobiliziranega encima, vpliv časa in temperature izpostavitve na ohranitev aktivnosti imobiliziranega ter prostega encima. Najvišjo učinkovitost imobilizacije smo dosegli po aktivaciji magnetnih nanodelcev z 1 % glutaraldehidom (GA), s koncentracijo encima 7,5 µL/mL ter časom imobilizacije 24 ur pri hitrosti stresanja 410 rpm. Kljub visoki učinkovitosti imobilizacije smo v večini primerov dobili zelo nizke ohranjene aktivnosti imobiliziranega encima, imobiliziran encim pa je pokazal boljšo stabilnost pri povišani temperaturi v primerjavi s prostim encimom. The purpose of the diploma thesis is the synthesis of magnetic maghemite nanoparticles coated with aminosilane and immobilization of lipase on a ready carrier. Maghemite nanoparticles were synthesized by the coprecipitation technique of ferrous II and ferric III ions with 25 % ammonia and coated with silica and afterwards with aminosilane. Process conditions for achieving the maximum efficiency and the residual activity of the immobilized enzyme were optimized, by changing the concentration of the enzyme, the type of a cross-linker, the time of immobilization and the concentration of a cross-linker glutaraldehyde. Our subject of interest was also the stability of the enzyme and how the remaining activity of the immobilized enzyme was affected by exposed time and temperature. Maximum efficiency was achieved after activating maghemite nanoparticles with 1 % glutaraldehyde (GA), with the enzyme concentration of 7,5 µL/mL after 24 hours at 410 rpm. Despite a good binding efficiency of the immobilization, very low residual activity was obtained in most cases. However, the immobilized enzyme showed a better stability at increased temperatures as compared to the wild enzyme.

Namen diplomske naloge je bila uspešna imobilizacija komercialnega encimskega preparata DENILITE II S na hitozanske maghemitne nanodelce in primerjava aktivnosti imobiliziranega encima s prostim encimom. Sintetizirali smo hitozanske maghemitne nanodelce po metodi kovalentne vezave in nanje imobilizirali encim. Za aktivacijo funkcionalnih skupin nosilca smo uporabili mrežna povezovalca glutaraldehid (GA) in pentaetilen heksamin (PEHA). Z različnimi koncentracijami mrežnega povezovalca smo aktivirali funkcionalne skupine nosilca ter iskali optimalno učinkovitost imobilizacije in preostalo aktivnost imobiliziranega encima. Zanimala nas je tudi stabilnost encima, kako vplivata čas in temperatura izpostavitve na ohranitev preostale aktivnosti imobiliziranega encima. Imobilizirani encim ima prednost pred prostim encimom, saj se lahko večkrat uporabi, zato smo preverili, kako vpliva večkratna uporaba na ohranitev preostale aktivnosti imobiliziranega encima. Najvišjo preostalo aktivnost imobiliziranega encima na hitozanske maghemitne nanodelce smo dosegli pri dodatku mrežnega povezovalca GA (3 %) in koncentraciji encima 0,05 g/mL. Encim, imobiliziran na hitozanske maghemitne nanodelce pri optimalnih pogojih, smo izpostavili za določen čas različnim temperaturam in opazili, da se preostala aktivnost ne ohrani. Proučevali smo tudi vpliv večkratne uporabe imobiliziranega encima na preostalo aktivnost. Imobiliziran encim smo večkrat ponovno uporabili in ugotovili, da aktivnost encima z naraščanjem števila ciklov upada. The goal of the thesis was to successfully immobilise DENILITE II S, a commercial enzyme preparation, onto maghemite nanoparticles coated by chitosan and to compare the activity of the immobilised enzyme to that of the free enzyme. The chitosan coated nanoparticles had been synthesised with the covalent bond method and the immobilised enzyme was added. To activate the functional groups of the carrier, glutaraldehyde (GA) and pentaethylenehexamine (PEHA) were used as crosslinking agents. By applying different concentrations of the crosslinking agents, functional groups of the carrier were activated and looked to determine the optimal efficiency of the enzyme immobilisation and the remaining activity of the immobilised enzyme. We were also interested in the stability of the enzyme and how the remaining activity of the immobilised enzyme was affected by exposed time and temperature. The immobilised enzyme has an advantage over the free enzyme, because it can be used more times therefore we examined how multiple usage affects the remaining activity of the immobilised enzyme. The highest values in the remaining activity of the enzyme immobilised onto maghemite nanoparticles coated by chitosan was achieved, when the crosslinking agent GA (3%) was applied and with enzyme concentration at 0.05 g/mL. The enzyme, immobilised onto chitosan coated nanoparticles under optimal conditions, was exposed to different temperatures for a certain period of time and we observed that the remaining activity does not remain preserved. The effect of multiple usage of the immobilised enzyme on its remaining activity was also examined. The immobilised enzyme was reused several times and the remaining activity of the enzyme decreased with the number of cycles.

Namen diplomskega dela je sinteza maghemitnih nanodelcev stabiliziranih s hitozanom in optimizacija pogojev za dosego najvišje učinkovitost imobilizacije in preostale aktivnosti imobiliziranega encima na tako pripravljene nosilce ter na biooglje pridobljeno s postopkom hidrotermične karbonizacije olivnih tropin in celuloze s sub-kritično vodo. Proučevali smo vpliv različnih parametrov na učinkovitost imobilizacije in preostalo aktivnost encima celulaze po končanem procesu imobilizacije na trde nosilce. S spreminjanjem pogojev smo želeli ugotoviti kateri nosilec je najbolj primeren za imobilizacijo celulaze. Spreminjali smo sledeče parametre: koncentracijo encima celulaze, čas imobilizacije, hitrost stresanja, vrsto mrežnega povezovalca ter koncentracijo mrežnega povezovalca. Najvišjo preostalo aktivnost imobiliziranega encima smo dosegli pri imobilizaciji encima na maghemitne nanodelce, prevlečene s slojem hitozana, pridobljene po drugem postopku oziroma po metodi zamreženja in aktiviranju nosilca z dodatkom 0,02 M pentaetilen heksamina (PEHA), pri koncentraciji encima 2,31 mg/mL, hitrosti stresanja 300 rpm in pri času imobilizacije 24 ur. The purpose of this thesis is the synthesis of the maghemite nanoparticles stabilized by chitosan and the optimization of conditions for achieving the maximum efficiency and the residual activity of the immobilized enzyme to this kind of carriers and biochar produced by hydrothermal carbonization of olive marc and cellulose in sub-critical water. By changing the conditions we observed how they affected the performance and activity of the enzyme of cellulase after the finished process of the immobilization of magnetic carriers. By changing the conditions we also wanted to determine which carrier was the most suitable for the immobilization of cellulase. We changed the concentration of the cellulase enzyme, the time of immobilization, the type and speed of shaking, the type and the concentration of cross-linker. Maximum activity was achieved in the case of immobilization of enzyme to maghemite nanoparticles coated with layer of chitosan after the second procedure (the method of cross-linking) and by activating the carrier with supplement of 0,02M pentatilen hexamine. We achieved the best conditions at a concentration of enzyme 2,31mg/mL, at the speed of shaking 300 rpm and at the time of immobilization 24hrs.

Namen diplomskega dela je sinteza magnetnih maghemitnih nanodelcev, prevlečenih s hitozanom po treh različnih postopkih in imobilizacija specifičnega biokatalizatorja, holesterol oksidaze, na tako pripravljen nosilec. Primerjali smo ohranjeno aktivnost encima holesterol oksidaze po končanem procesu imobilizacije in učinkovitost imobilizacije encima na maghemitne nanodelce, prevlečene s hitozanom po treh različnih postopkih. S spreminjanjem procesnih pogojev smo želeli ugotoviti kateri delci bi bili najprimernejši za imobilizacijo encima holesterol oksidaze. Spreminjali smo koncentracijo mrežnega povezovalca glutaraldehida, hitrost stresanja in vrsto stresanja pri imobilizaciji encima, vrsto mrežnega povezovalca in koncentracijo encima holesterol oksidaze. Zanimala nas je tudi stabilnost imobiliziranega encima v primerjavi s prostim encimom in vpliv večkratne uporabe imobiliziranega encima na ohranjeno aktivnost encima. Najvišjo ohranjeno aktivnost in učinkovitost smo dosegli pri imobilizaciji encima na maghemitne nanodelce, prevlečene s hitozanom po tretjem postopku (metoda kovalentne vezave) in dodatku mrežnega povezovalca PEHA (0,02 M). Najboljše pogoje smo dosegli pri koncentraciji encima 1 mg/mL, pri hitrosti stresanja 300 rpm in času imobilizacije 24 ur. Tako imobiliziran encim smo lahko ponovno uporabili in z njim smo izvedli deset ciklov. Po 122 urah pa encim ni bil več aktiven. The purpose of the diploma thesis is the synthesis of magnetic maghemite nanoparticles coated with chitosan after three different procedures and immobilization of a specific biocatalyst, cholesterol oxidase, on a ready carrier. We compared the preserved activity of the enzyme cholesterol oxidase after the immobilization process and the efficiency of enzyme immobilization on maghemite nanoparticles coated with chitosan after three different procedures. By varying process conditions, we wanted to determine which particles would be most suitable for immobilization of the enzyme cholesterol oxidase. We changed the concentration of a crosslinker glutaraldehyde, the speed of shaking and the type of shaking at the immobilization of an enzyme, the type of a crosslinker and the concentration of the enzyme cholesterol oxidase. We were interested in the stability of the immobilized enzyme in comparison to the free enzyme, and the influence of the multiple use of an immobilized enzyme on a preserved activity of an enzyme. We have achieved the highest preserved activity and efficiency at the immobilization of an enzyme on the maghemite nanoparticles, coated with chitosan, after the third procedure (the method of covalent bonding) and with the addition of a crosslinker PEHA (0,02 M). We have achieved the best conditions were at a concentration of an enzyme 1 mg/mL, at a shaking speed of 300 rpm and an immobilization for 24 hours. Thus immobilized enzyme could we re-use for ten cycles. After 122 hours enzyme was no longer active.

Namen diplomske naloge je optimizacija procesnih pogojev za imobilizacijo encima hrenove peroksidaze na površinsko modificirane maghemitne nanodelce. Spreminjali smo masno razmerje med nosilcem in encimom, koncentracijo mrežnega povezovalca glutaraldehida in čas imobilizacije. Primerjali smo učinkovitost imobilizacije ter ohranjeno aktivnost encima, vezanega na aminosilanske in hitozanske maghemitne nanodelce. Maghemitne nanodelce smo sintetizirali s koprecipitacijo Fe2+ in Fe3+ ionov ob dodatku obarjalnega reagenta, 25 % amonijaka. Aminosilanske maghemitne nanodelce smo pripravili v dveh stopnjah. Najprej smo nanodelce prevlekli s funkcionalno plastjo silike (SiO2), nato pa še s plastjo aminosilana. Hitozanski maghemitni nanodelci so bili pripravljeni po treh različnih postopkih. Kljub dobri učinkovitosti imobilizacije, se v nobenem primeru ni ohranila visoka aktivnost encima. Najvišjo ohranjeno aktivnost imobiliziranega encima smo dosegli pri aminosilanskih nanodelcih, in sicer 2,06 % pri 65,76 % učinkovitosti imobilizacije. Prav tako je imobiliziran encim v primerjavi s prostim encimom pokazal nižjo stabilnost pri povišanem tlaku in temperaturi. Z zvišanjem temperature pri atmosferskem tlaku, se je aktivnost prostega in imobiliziranega encima izboljšala. The purpose of the diploma thesis is optimization of process conditions for immobilization of horseradish peroxidase onto surface modified maghemite nanoparticles. By varying the mass ratio between carrier and enzyme, concentration of glutaraldehyde and duration time of immobilization, the comparison of the binding efficiency and residual activity of enzyme, immobilized onto maghemite nanoparticles coated by aminosilane and chitosan was done. Maghemite nanoparticles were synthetized by coprecipitation of Fe2+ and Fe3+ ions by adding an 25 % ammonia as precipitation agent. Aminosilane coated maghemite nanoparticles were prepared in two stages. First, maghemite nanoparticles were functionalized with silica (SiO2) and afterwards with aminosilane. Chitosan coated nanoparticles were prepared by three different procedures. Despite well binding efficiency, there were no high residual activities obtained. The highest activity preserved was in case of enzyme, immobilized onto aminosilane coated nanoparticles: 2,06 % at 65,76 % binding efficiency. Immobilized enzyme also demonstrated lower stability at higher pressure and temperature in comparison with free enzyme. Residual activity of free and immobilized enzyme increased by raising temperature at atmospheric conditions.