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Halogenhidrin-dehalogenaza tipa C (HheC iz Agrobacterium radiobacter) zauzima važno mjesto u industrijskoj biokatalizi zahvaljujući visokoj enantioselektivnosti i mogućnosti sinteze raznovrsnih β-supstituiranih alkohola i epoksida [1]. Zbog hidrolitičke nestabilnosti i ograničene topljivosti supstrata epoksida, HheC-katalizirane reakcije mogu se provoditi u alternativnim medijima poput organskih otapala. Pritom otapala mogu imati štetan utjecaj na strukturna i katalitička svojstva enzima. Stoga se operacijska stabilnost enzima može povećati metodama imobilizacije poput kapsuliranja u metalo-organske mreže (engl. metalorganic frameworks – MOFs). MOF su porozni materijali visokog stupnja kristaliničnosti koji mogu zaštititi enzim od nepovoljnih reakcijskih uvjeta. Zeolitne imidazolne mreže (ZIF) moguće je sintetizirati pri biokompatibnilim uvjetima gdje enzim služi kao nukleus kristalizacije [2]. U ovom radu ispitana je mogućnost sinteze HheC@ZIF-8 s ciljem povećanja stabilnosti prilikom biokatalize u organskom mediju. Ovaj rad izrađen je u okviru projekta „Enzimska sinteza fluoriranih kiralnih građevnih blokova“ EnzyFluor (IP- 2018-01-4493) financiranog sredstvima Hrvatske zaklade za znanost te hrvatskoslovenskog bilateralnog projekta „Stabilizacija halogenhidrin-dehalogenaza radi upotrebe u nekovencionalnim medijima“.

Halohydrin dehalogenases (HHDHs) are important and versatile biocatalysts, that can be used for the synthesis of important building blocks and precursors in the pharmaceutical, agrochemical and chemical industries. HHDHs have a wide spectrum of different compounds, such as chiral epoxides, β-substituted alcohols, oxazolidinones and others, that can be synthesized by their action. However, the enzyme was reported to lack stability under operational conditions. Therefore, immobilization of HHDH onto magnetic nanoparticles (MNPs) represents a powerful tool to overcome its limitations and improve its enzyme characteristics as a biocatalyst. HHDH was immobilized onto bare maghemite nanoparticles, chitosan coated MNPs and carboxymethyl dextran (CMD) coated MNPs. All types of MNPs were prepared by co-precipitation method of ferrous and ferric ions. The immobilization protocol was initially optimized using 10% (v/v) cross-linker glutaraldehyde (GA) in order to improve residual activity and immobilization efficiency of immobilized HHDH. The highest immobilization efficiency (91%) of immobilized HHDH was achieved, while using CMD coated MNPs, which were cross-linked with 10% (v/v) GA for 1 hour at 350 rpm and later immobilized with HHDH for 1 hour at 350 rpm. To implement HHDH at industrial scale, initial optimization of HHDH immobilization was performed, which offers room for improvement in order to achieve an effective biocatalyst for various synthetic applications.

Doktorska disertacija zajema dva dela, v prvem delu smo se osredotočili na sintezo magnetnega nosilca, modificiranega z organskim polimerom karboksimetil dekstranom (CMD). V sintezni postopek smo vpeljali tri različne koncentracije CMD (0,25 g/mL, 0,40 g/mL in 0,50 g/mL CMD) ter sintetizirali tri različne modificirane magnetne nanodelce (CMD1-MNPs, CMD2-MNPs in CMD3-MNPs). Sintetizirane CMD-MNPs smo okarakterizirali z različnimi analiznimi metodami: Fourier-transformirano infrardečo spektroskopijo (FT-IR), termogravimetrično analizo (TGA), vrstično elektronsko mikroskopijo (SEM), energijsko disperzijsko spektroskopijo (EDS), transmisijsko elektronsko mikroskopijo (TEM), z meritvami dinamičnega sipanja svetlobe (DLS). Magnetne lastnosti smo določili z elektronsko paramagnetno resonanco (EPR) in vibracijskim magnetometrom (VSM). Uspešno smo sintetizirali magnetne nosilce CMD-MNPs z ozkimi porazdelitvami nanovelikosti od 27-30 nm. S FT-IR analizo smo določili prisotnost karboksilnih in hidroksilnih skupin na površini CMD-MNPs, kar potrjuje prisotnost polimerne prevleke CMD na sintetiziranih MNPs. Z meritvami EPR in VSM smo dokazali, da imajo sintetizirani CMD-MNPs magnetne lastnosti ter feromagnetni sistem. Določili smo inhibitorne lastnosti CMD-MNPs na rast dveh bakterijskih kultur. Inhibitorne učinke na rast testnih mikroorganizmov smo zaznali pri CMD3-MNPs, medtem ko ostali CMD-MNPs in neprevlečeni MNPs ne izkazujejo antimikrobne učinkovitosti. Proučevali smo tudi inhibitorne lastnosti MNPs modificiranih s hitozanom (HIT-MNPs) in aminosilanom (AMS-MNPs) na petih različnih bakterijskih kulturah, pri katerih nismo zaznali inhibiornega učinka na rast izbranih mikororganizmov. Za nadaljnje raziskave smo izbrali CMD3-MNPs, na katerega smo vezali encim alkohol dehidrogenazo (ADH). V drugem delu doktorske disertacije smo nosilec CMD3-MNPs površinsko funkcionalizirali z epiklorohidrinom (EClH). Optimalna koncentracija EClH je znašala 4 % (v/v). Proučevali smo vpliv različnih procesnih parametrov na preostalo aktivnost in učinkovitost imobilizacije ADH na CMD3-MNPs. Pri optimalnih pogojih imobilizacije ADH na CMD3-MNPs smo dosegli 89,6 % preostalo aktivnost imobilizirane ADH in 99,5 % učinkovitost imobilizacije. Nadaljevali smo s postopkom koimobilizacije, pri čemer smo na funkcionaliziran nosilec CMD3-MNPs koimobilizirali encim ADH in kofaktor β-nikotinamid adenin dinukleotid (β-NAD). S spreminjanjem procesnih parametrov smo proučevali njihov vpliv na preostalo aktivnost imobilizirane ADH s kofaktorjem β-NAD na CMD3-MNPs in učinkovitost koimobilizacije. Pri optimalnih pogojih koimobilizacije ADH in β-NAD na CMD3-MNPs smo dosegli 73,3 % preostalo aktivnost ADH ter 93,8 % učinkovitost imobilizacije. Izvedli smo še študijo termične stabilnosti proste ADH, ADH imobilizirane na CMD3-MNPs in ADH koimobilizirane z β-NAD na CMD3-MNPs pri različnih temperaturah. ADH imobilizirana na CMD3-MNPs je ohranila skoraj 60 % svoje začetne aktivnosti po 24. urah inkubacije pri temperaturah 20 °C in 40 °C. ADH koimobilizirana z β-NAD na CMD3-MNPs je pri temperaturi 30 °C ohranila 75,4 % začetne aktivnosti, pri 50 °C pa 66,5 % začetne aktivnosti po 5. urah inkubacije. Proučili smo še stabilnost ADH imobilizirane na CMD3-MNPs in ADH koimobilizirane z β-NAD na CMD3-MNPs, ki smo ju skladiščili pri 4 °C. Po treh tednih sta obe obliki imobilizrane ADH ohranili 60 % svoje začetne aktivnosti. Magnetne nosilce HIT-MNPs in AMS-MNPs smo funkcionalizirali z mrežnim povezovalcem glutaraldehidom (GA) in amino-donorjem pentaetilenheksaminom (PEHA), na katere smo imobilizirali encim β-galaktozidazo (β-GAL) ter optimirali koncentraciji GA in PEHA. Pri kombinaciji obeh GA in PEHA smo dosegli hiperaktivacijo encima (128,9 %), do katere pride zaradi konformacijskih sprememb encima. Hiperaktivacijo smo dosegli tudi pri imobilizaciji β-GAL na AMS-MNPs. Najvišjo preostalo aktivnost β-GAL smo dosegli, kadar smo kot mrežni povezovalec uporabili 20 % (v/v) PEHA (154,4 %). Doctoral thesis consists of two parts, in the first part synthesis of magnetic carrier and modification with organic polymer carboxymethyl dextran (CMD) was performed. Three different CMD concentrations were applied into the synthesis process (0,25 g/mL, 0,40 g/mL in 0,50 g/mL CMD), which resulted in three different modified magnetic carriers (CMD1-MNPs, CMD2-MNPs and CMD3-MNPs). All CMD-MNPs were characterized with various analytical methods: Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDX), transmission electron microscopy (TEM), dynamic light scattering (DLS). Magnetic properties were analyzed with electron paramagnetic resonance (EPR) and magnetization measurements with vibrating sample magnetization (VSM). CMD-MNPs were successfully synthesized with average diameters from 27-30 nm and size distribution revealed most consistent sizes of CMD3-MNPs. Hydroxyl and carboxyl groups were confirmed on the CMD-MNPs surface, which confirms the presence of CMD layer on the surface of CMD-MNPs. EPR and VSM measurements confirmed magnetic properties of all CMD-MNPs and a ferromagnetic system. Inhibition properties were determined of all CMD-MNPs on two different bacterial cultures, where CMD3-MNPs displayed inhibition zone, confirming antimicrobial properties, whereas with other CMD-MNPs no inhibition was detected. Toxicity was also determined on MNPs coated with chitosan (HIT-MNPs) and aminosilane (AMS-MNPs) on five different bacterial cultures, where no inhibition was detected. CMD3-MNPs was chosen for further investigation of enzyme immobilization in the second part of doctoral thesis, because of its most favourable properties. In second part of doctoral thesis nano-carrier CMD3-MNPs was surface functionalized with epoxy cross-linking, using epoxy cross-linker epichlorohydrin (EClH) to covalently bind enzyme alcohol dehydrogenase (ADH). With optimization of process parameters EClH with 4 % (v/v) was used, which resulted in the highest residual activity of immobilized ADH. Epoxy-functionalized CMD3-MNPs were used in immobilization protocol, where effect of process parameters was investigated on the residual activity and immobilization efficiency of ADH. After successful optimization ADH immobilized onto CMD3-MNPs managed to obtain 89,6 % of residual activity and 99,5 % of immobilization efficiency. Further on, co-immobilization of enzyme ADH and cofactor β-nicotinamide adenine dinucleotide (β-NAD) was performed. Again, effect of process parameters on residual activity and immobilization efficiency of co-immobilization were investigated. After successful optimization, ADH co-immobilized with β-NAD onto CMD3-MNPs managed to obtain 73,3 % of residual activity and 93,8 % of immobilization efficiency. Thermal stability of immobilized ADH at different temperatures was investigated. ADH immobilized onto CMD3-MNPs obtained almost 60 % of its initial activity after 24 hours at 20 °C and 40 °C, ADH co-immobilized with β-NAD onto CMD3-MNPs managed to obtain 75,4 % of its initial activity at 30 °C and 66,5 % of its initial activity at 50 °C after 5 hours. Storage stability of ADH immobilized onto CMD3-MNPs and ADH co-immobilized with β-NAD onto CMD3-MNPs were investigated at 4 °C, where both obtained almost 60 % of its initial activity after three weeks. Magnetic carriers HIT-MNPs and AMS-MNPs were functionalized with cross-linker glutaraldehyde (GA) and amino-donor pentaethylenehexamine (PEHA). Functionalized HIT-MNPs and AMS-MNPs were immobilized with enzyme β-galactosidase (β-GAL). When using combination of 0,5 % (v/v) GA and 30 % (v/v) PEHA 128,9 % of residual activity was achieved, which resulted in hyper-activation of enzyme due to its conformational changes. Hyper-activation was achieved also with immobilizing β-GAL onto AMS-MNPs and the highest residual activity was obtained with with 20 % (v/v) of PEHA (154,4 %).

Nanoparticles of inorganic magnetic core surrounded by layers of functional coatings are potential representatives of nanostructures for immobilization of bio-substances. Magnetic nanoparticles (MNPs) are often bound in aggregates due to a strong magnetic dipole, which has a lot of advantages, such as large surface area for binding biologically active substances. Chitosan is a polysaccharide polymer that is non-toxic, hydrophilic, biocompatible and has hydroxy and amino groups in its structure. Because of these chemical and biological properties it is a desirable bio-product for immobilization of enzymes and for binding of other biologically active substances. Magnetic micro and nanoparticles were synthesized with chitosan by three different methods microemulsion process, suspension cross-linking technique and covalent binding of chitosan. Toxic effect of the prepared magnetic particles was determined as well and was examined on five different bacterial cultures Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae. At concentrations of 10-30 mg of magnetic particles per 0.5 McFarland Standard solution of E. coli and per 400 CFU of S. aureus, P. aeruginosa, E. faecalis in K. pneumonia, no inhibition on the chosen bacterial cultures was detected. Nanodelci iz magnetnega anorganskega jedra, oblečeni z večslojno funkcionalno prevleko, predstavljajo pomemben razred nanostrukturiranih delcev za vezavo biosubstanc. Zaradi možnega magnetnega dipola se magnetni nanodelci pogosto združujejo v skupke, kar pa ima številne prednosti, kot na primer velika površina, na katero lahko pritrdimo različne biološke komponente. Hitozan je polisaharidni polimer, ki je nestrupen, hidrofilen, biokompatibilen in vključuje prisotnost hidroksilne in amino skupine v svoji strukturi. Zaradi naštetih kemijskih in bioloških lastnosti spada hitozan med zaželjene biomateriale za imobilizacijo encimov in vezavo drugih biološko aktivnih substanc. Magnetne nanodelce, prevlečene s hitozanom, smo pripravili po treh različnih postopkih s postopkom mikroemulzije, s postopkom suspenzijske zamreževalne tehnike ter s postopkom kovalentne vezaven hitozana. Toksikološke vplive pripravljenih magnetnih delcev smo preverili na petih različnih bakterijskih kulturah Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis in Klebsiella pneumoniae. Pri koncentracijah 10-30 mg magnetnih delcev na 0.5 McFarland standardne raztopine bakterijske kulture E.coli in na 400 CFU bakterijskih kultur S. aureus, P. aeruginosa, E. faecalis in K. pneumonia do inhibicije rasti mikroorganizmov ni prišlo.

Diplomsko delo opisuje določevanje aktivnosti nekaterih encimov v različnih vzorcih granatnega jabolka. Vzorci, v katerih smo določevali encimsko aktivnost so naslednji: sveži sok, liofilizirani sok, zamrznjeni sok, etanolni ekstrakt svežih pečk, etanolni ekstrakt svežih lupin, etanolni ekstrakt liofiliziranih pečk, etanolni ekstrakt liofiliziranih lupin, vodni ekstrakt svežih pečk, vodni ekstrakt svežih lupin in vodni ekstrakt liofiliziranih lupin.Vzorce smo pridobili z različnimi postopki, kot so centrifugiranje, homogeniziranje in ekstrakcija z etanolom. V teh vzorcih smo določevali tudi totalne fenole, proantocianide, antioksidativnost, vitamin C in vitamin E. Encimi, katere smo določevali v naših vzorcih so: α-amilaza, celulaza, katalaza, lipaza, peroksidaza, proteaza in transglutaminaza. Z Bradfordovo metodo smo vzorcem določili koncentracijo proteinov. Granatno jabolko je polno vitaminov in fenolov, kar smo z rezultati tudi dokazali, saj smo v vseh vzorcih dobili prisotne večje količine vitaminov in fenolov. Prav tako so rezultati pokazali, da so prisotni vsi encimi, katere smo določali, vendar ne v vseh vzorcih. The diploma paper describes the determination of the activity of certain enzymes in different samples of pomegranate apples. The samples in which the enzyme activity was determined are as follows: fresh juice, lyophilized juice, frozen juice, ethanol extract of fresh stoves, ethanol extract of fresh peel, ethanol extract of lyophilized pellets, ethanol extract of lyophilized shells, aqueous fresh stoves extract, fresh peel extract and an aqueous extract of lyophilized shells. The samples have been obtained by various processes such as centrifugation, homogenization and extraction with ethanol. Relatively high proportion of total phenols, proanthocyanides, antioxidants, vitamin C and vitamin E has been demonstrated. The identified enzymes in our samples are following: α-amylase, cellulase, catalase, lipase, peroxidase, protease and transglutaminase. The Bradford method was used to determine the concentration of proteins in the samples. Pomegranate apple is wholesome fruit with a high content of vitamins and phenols. Their presencehas been proved by the obtained results, since high amounts of vitamins and phenols was determined in all of samples. The results also indicate the presence of all determined enzymes , but not in all samples.