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Optički čisti spojevi od velike su važnosti u farmaceutskoj indrustriji, uz sve veći interes za fluorirane spojeve. Za sintezu kiralnih gradivnih elemenata često se koriste enzimi halogenhidrin-dehalogenaze (HHDH), koje mogu primiti brojne anionske nukleofile, od cijanida, cijanata, tiocijanata do azida i nitrita. U njihovoj prisutnosti dolazi do reakcije otvaranja epoksidnog prstena, što dovodi do stvaranja β-substituiranog alkohola. Osnova ovog rada je biokatalitička reakcija, točnije HHDH-katalizirana reakcija otvaranja epoksidnog prstena rac-2-(4-fluorofenil)oksirana cijanidnim ionima. Uz željeni produkt (S)-3-(4-fluorofenil)-3-hidroksipropannitril, ovom reakcijom stvara se i neželjeni (R)-3-(4-fluorofenil)-3-hidroksipropannitril te rac-2-(4-fluorofenil)-1,2-etandiol. Prethodno je razvijen matematički model ove sinteze kombinacijom kinetičkih jednadžbi i reaktorskog modela. U svrhu validacije modela, provedena je serija eksperimenata u kotlastom reaktoru s različitim početnim koncentracijama epoksida, NaCN i HHDH. U eksperimentima inkubacije uočena je deaktivacija enzima ovisna o početnoj koncentraciji epoksida. Operacijska stabilnost matematički je opisana i uključena u model. Nadalje, porastom koncentracije epoksida, stabilnost enzima opada, dok cijanidni nukleofil nema značajan utjecaj na stabilnost enzima. Eksperimentalni podaci pokazuju zadovoljavajuće slaganje s ranije razvijenim matematičkim modelom. Također, matematičko modeliranje pokazalo je kako je sinteza u kotlastom reaktoru/kotlastom reaktoru s ponavljajućim dodatkom epoksida (repetitivnom šaržnom reaktoru) optimalan način proizvodnje danog spoja. Optically pure compounds are of great significance in the pharmaceutical industry, with the increasing interest in fluorinated compounds. One important enzyme group often employed in synthesizing various chiral building blocks are halohydrin dehalogenases (HHDHs) that can accept numerous anionic nucleophiles from cyanide, cyanate, thiocyanate to azide and nitrite. In their presence the ring-opening reaction of epoxide occurs, leading to β-substituted alcohol formation. The foundation of this work lies in a biocatalytic reaction, precisely HHDH-catalysed rac-2-(4-fluorophenyl)oxirane ring-opening reaction mediated by cyanide ions. Besides the desired (S)-3-(4-fluorophenyl)-3-hydroxypropanenitrile, this reaction yields an undesired (R)-3-(4-fluorophenyl)-3-hydroxypropanenitrile and rac-2-(4-fluorophenyl)-1,2-ethanediol. Mathematical model was earlier developed by coupling kinetic equations with reactor model. A series of different batch experiments varying in initial concentrations of epoxide, NaCN and HHDH were conducted with aim of model validation. Enzyme deactivation influenced by the initial epoxide concentration was observed within incubation experiments. Operational stability decay was mathematically described and included in the model. Furthermore, with the increase in the epoxide concentration, enzyme stability decreased, while the cyanide nucleophile did not have a significant effect on the stability of the enzyme. Experimental data has shown a satisfactory match with an earlier developed mathematical model. Moreover, mathematical modeling has shown that synthesis in batch/repetitive batch reactor is the optimal pathway for production of this compound.

Ovaj diplomski rad sastoji se od dva dijela: istraživačkog i metodičkog. U istraživačkom dijelu rada pročišćene su tri varijante enzima adenilosukcinat-sintetaze (AdSS) iz bakterije Helicobacter pylori: AdSS obilježen histidinskim privjeskom, AdSS kojem je aspartat 12 zamijenjen alaninom i AdSS kojem je aspartat 12 zamijenjen asparaginom. Za pročišćavanje su korištene kromatografija ionske izmjene, afinitetna kromatografija i gel filtracija. Proteinima su potom mjerene specifične aktivnosti, a za obiljeţen AdSS određeni su kinetički parametri uz promjenjivu koncentraciju iona Mg2+ koristeći Michaelis-Menten model enzimske kinetike. Konačno, ispitan je utjecaj različitih iona metala na aktivnost obilježenog AdSS. U metodiĉkom dijelu rada pod naslovom „Priča o želatini i voću“ izrađena je metodičkodidaktička priprema za učenike četvrtog razreda gimnazije. Metodička priprema temeljena je na obrazovnim ishodima, najčešćim učeničkim krivim shvaćanjima i na uvodu u relevantnu literaturu. Pripremljeni materijal namijenjen je obrazovnoj strategiji učenja otkrivanjem. This Diploma thesis consists of two parts: the research and methodological part. In the experimental part of diploma work, three variants of enzyme adenylosuccinate synthetase (AdSS) from bacterium Helicobacter pylori were purified: AdSS with histidine tag, AdSS with aspartate 12 changed to alanine and AdSS with aspartate 12 changed to asparagine. Ion exchange, affinity and gel filtration chromatographies were used for purification. Specific activity was measured for each enzyme variant, and for tagged AdSS kinetic parameters were also determined, by changing Mg2+ ions concentration, and using Michaelis-Menten model of enzyme kinetics. Finally, the effect of different metal ions on tagged AdSS activity was investigated. In the methodological part under the title “The story of gelatin and fruit” a didacticmethodological preparation was made for fourth grade high school students. The methodological preparation is based on the educational outcomes, common misconceptions held by students and with an insight in a revelant literature. The prepared material is intended for educational strategy of learning by discovery.

Enzimske kaskadne reakcije predstavljaju obećavajući način sinteze tvari zbog većih iskorištenja procesa i smanjenja potrebe za dodavanjem drugih komponenti u proces te za pročišćavanjem produkata reakcije. Matematički modeli mogu dati dobar i brzi uvid u ovakve procese bez potrebe za provođenjem prevelikog broja eksperimenata. U ovom radu je razvijen matematički model sinteze ATP-a u kaskadnoj reakciji te su provedene simulacije pri različitim koncentracijama adenozina i magnezija. Time su dobiveni uvjeti pri kojima je moguće postići najveću koncentraciju ATP-a. Također je korištenjem razvijenog modela procijenjena osjetljivost kinetičkih parametara svake pojedinačne reakcije u ovom kaskadnom procesu. Prema dobivenim rezultatima može se zaključiti da parametri napredne reakcije katalizirane enzimom PPK2-I najviše utječu na ishod kaskadne reakcije. Enzymatic cascade reactions are a promising way to synthesize substances due to the higher yield and the reduction of the need to add other components to the process and to purify the reaction products. Mathematical models can give a good and quick insight into such processes without the need to conduct too many experiments. A mathematical model of ATP synthesis in a cascade reaction was developed according to the obtained experimental data and simulations of the cascade reaction at different concentrations of adenosine and magnesium were performed. In that way conditions under which it is possible to achieve the highest concentration of ATP were estimated. Parameter sensitivity of each reaction in the cascade was estimated by simulations using the mathematical model. According to the obtained results it can be concluded that parameters in the forward reaction catalyzed by the enzyme PPK2-I have the highest effect on the outcome of the cascade reaction.

Adenilosukcinat-sintetaza (AdSS) je esencijalan enzim u biosintezi purina, i de novo, i reciklirajućim putem. U ovoj disertaciji je računalno i eksperimentalno karakterizirana AdSS iz bakterije Helicobacter pylori koja pripada skupini I kancerogena. Pročišćen je enzim sa i bez histidinskog privjeska na C-kraju. Za oba enzima su određeni specifična aktivnost, temperaturno i pH područje stabilnosti, te kinetički parametri prema supstratima i inhibitorima. Dobiveni rezultati ukazuju na to da dodani histidinski privjesak nema značajan utjecaj na funkciju enzima. Riješena kristalna struktura AdSS pri visokoj rezoluciji, korištena za karakterizaciju enzima računalnim metodama, otkriva stvaranje stabilnog međuprodukta 6'-fosforil-IMP u aktivnom mjestu enzima. Računalnim metodama identificirane su ključne interakcije za stabilizaciju supstrata u aktivnom mjestu. Analiza kristalne strukture i računalna karakterizacija interakcija proteina s ligandima ukazuju na veliku sličnost AdSS iz bakterije H. pylori s AdSS iz vrste Escherichia coli, stoga se može pretpostaviti i sličan mehanizam rada enzima. Adenylosuccinate synthetase (AdSS) is an essential enzyme in de novo and recycling pathway of purine biosynthesis. In this thesis, AdSS from bacterium Helicobacter pylori, a group I carcinogen, was characterized experimentally and computationally. Enzyme with and without histidine tag at the C-terminus was purified. Specific activity, temperature and pH stability range, and kinetic parameters toward substrates and inhibitors were determined for both enzymes. Obtained results indicate that the added histidine tag has no significant effect on enzyme function. Solved crystal structure at high resolution, used to characterize the enzyme computationally, reveals the formation of a stable intermediate 6'-phosphoryl-IMP in the active site. Computational methods were used to identify key interactions for stabilisation of substrates in the active site. Analysis of crystal structure and computational characterization of protein-ligand interactions indicate high similarity of H. pylori AdSS to Escherichia coli AdSS, therefore the similar molecular mechanism for this enzyme can be assumed.

Flavonoidi su velika i heterogena skupina polifenolnih spojeva niske molekulske mase koje ljudi hranom svakodnevno unose, a organizmu predstavljaju ksenobiotike kojih se nastoji riješiti. Među najvažnijim mehanizmima koje ljudski organizam ima na raspolaganju za eliminaciju ksenobiotika jest oksidativni metabolizam koji je najvećim dijelom posredovan citokromima P450. Iako su rezultati istraživanja bioloških učinaka in vitro obećavajući, flavonoidi često ne ostvaruju svoj farmakološki potencijal in vivo zbog slabe bioraspoloživosti. Fiziološki mehanizmi djelovanja i metabolička sudbina flavonoida još su uvijek vrlo slabo istraženi. Stoga je cilj ovoga rada bio karakterizirati oksidativni metabolizam većeg broja odabranih flavonoida posredovan humanim citokromima P450. U tu svrhu ispitano je 30 odabranih aglikona flavonoida u inkubacijama s humanim jetrenim mikrosomima i rekombinantnim citokromima P450. Nastanak metabolita praćen je tekućinskom kromatografijom spregnutom sa spektrometrijom masa visoke razlučivosti. U 15 od 30 analiziranih flavonoida primijećen je metabolizam posredovan citokromima P450 1A2, 2C19, 2D6, 2E1 i/ili 3A4. Detektirani metaboliti nastali su reakcijama aromatske hidroksilacije ili O-demetilacije ili kombinacijom ovih reakcija. Vrijednosti konstante specifičnosti (kcat/Km) svih primijećenih metaboličkih reakcija kretale su se u širokom rasponu (0,0015-2,9) × 106 M–1 min–1 što upućuje na visoku selektivnost humanih jetrenih citokroma P450 prema flavonoidima. Enzim koji je daleko najučinkovitije metabolizirao ispitane flavonoide bio je CYP1A2 što sugerira da je zbog mogućih interakcija potreban dodatni oprez kod unosa flavonoida i istovremene primjene lijekova koji se primarno metaboliziraju putem CYP1A2. U određenoj su mjeri opisana i jedinstvena strukturna obilježja odgovorna za metabolizam posredovan citokromima P450 koja omogućuju predviđanje mogućih metaboličkih putova i za flavonoide koji nisu bili predmetom ovog istraživanja. Dobiveni rezultati omogućuju bolje razumijevanje metabolizma flavonoida što može pomoći u prevladavanju niske bioraspoloživosti flavonoida in vivo i usmjeriti napore istraživača prema pronalaženju flavonoida s povoljnim terapeutskim i metaboličkim profilom. Flavonoids are large and heterogeneous group of polyphenolic compounds with low molecular mass that people ingest in everyday diet, but represent xenobiotics which human organism tends to eliminate. Among most important mechanisms that human body has at disposal for elimination of xenobiotics is oxidative metabolism mainly mediated by cytochromes P450. Although results of in vitro studies are promising, due to low bioavailability flavonoids often do not demonstrate their pharmacological potential in vivo. Physiological mechanisms of act as well as metabolic fate of flavonoids are still poorly understood. The objective of this work was to characterize oxidative metabolism mediated by human cytochromes P450 of larger number of flavonoid compounds. For that purpose, 30 selected flavonoid aglycones were examined in incubations with human liver microsomes and recombinant cytochromes P450. The formation of metabolites was monitored by liquid chromatography coupled with high resolution mass spectrometry. Out of 30 analyzed flavonoids, 15 was susceptible to oxidative metabolism mediated by cytochromes P450 1A2, 2C19, 2D6, 2E1 and/or 3A4. The detected metabolites were formed by aromatic hydroxylation or O-demethylation, or combination of these reactions. The values of catalytic effectiveness (kcat/Km) of all observed metabolic reactions were obtained in very wide range from 0.0015 to 2.9 × 106 M–1 min–1 which indicates high selectivity of human liver cytochromes P450 to flavonoids. The dominant enzyme responsible for the observed metabolic reactions was CYP1A2 suggesting that potential interactions of flavonoids with drugs primarily metabolized by CYP1A2 should be considered. Specific structural characteristics responsible for metabolism mediated by cytochromes P450 were determined to some extent thus enabling prediction of possible metabolic pathways for flavonoids other than those analyzed in this study. These results contribute to better understanding of flavonoid metabolism thus helping to overcome their low bioavailability in vivo and steer the efforts of researchers towards detecting flavonoids with desirable therapeutic and metabolic profiles.

Statini su skupina lijekova koja se koristi kako bi se snizila razina kolesterola u krvotoku čovjeka s ciljem sprječavanja razvoja kardiovaskularnih bolesti. Statini posjeduju 3,5-dihidroksi kiselinski bočni lanac s dva kiralna središta koji su zaslužni za ostvarivanje glavne funkcije lijeka. Radi velikih zahtijeva kemijske i stereokemijske čistoće, njihova sinteza predstavlja izazov. U ovom radu provedena je biokatalitička oksidacija α-kloroaldehida (laktola) u aktivni kiselinski oblik bočnog lanca statina katalizirana oksidoreduktazama. Kao oksidoreduktaze korišteni su enzimi aldehid dehidrogenaza (AlDH) i ketoreduktaza (KRED). Obzirom da navedeni enzimi za svoju aktivnost trebaju prisutnost koenzima NAD(P)+ provedena je njegova regeneracija enzimom NAD(P)H oksidazom (NOX). Ispitana je kinetika svih enzima. Oksidacija laktola uz AlDH opisana je dvosupstratnom Michaelis-Menteničinom kinetikom s uključenom inhibicijom supstratom, NAD+, te kompetitivnom inhibicijom produktom, NADH. Oksidacija laktola uz KRED opisana je dvosupstratnom Michaelis-Menteničinom kinetikom s uključenom inhibicijom supstratom, NADP+, te kompetitivnom inhibicijom produktom, NADPH. Kinetika enzima NOX u reakciji oksidacije NADPH opisana je Michaelis-Menteničinom kinetikom s kompetitivnom inhibicijom produktom (NADP+). Pored navedenih kinetičkih mjerenja ispitan je i utjecaj aldehida na stabilnost enzima AlDH, KRED i NOX-a. Razvijen je matematički model procesa te je validiran provedbom reakcije u kotlastom reaktoru. Provedeni su eksperimenti oksidacije pročišćenog laktola s navedenim enzimima uz regeneraciju koenzima, te sirovog laktola dobivenog aldolnom adicijom acetaldehida i kloroacetaldehida katalizirane enzimom 2-deoksiriboza-5-fosfat aldolazom (DERA). Statins are group of drugs used for lowering cholesterol level in human bloodstream in order to prevent cardiovascular diseases. Statins possess 3,5-dihydroxy acid side chain with two chiral canters which are responsible for achieving the main function of the drug. Synthesis of statin side chains is a challenge due to high demands for chemical and stereochemical purity. In this work a biocatalytical oxidation of α-chloroaldehyde (lactol) was carried out catalyzed by oxidoreductases in order to produce the statin side chain. For this purpose, enzymes aldehyde dehydrogenase (AlDH) and ketoreductase (KRED) were tested. Since the oxidoreductases for its activity demand the use of coenzyme NAD(P)+ which need to be regenerated, the enzyme NAD(P)H oxidase (NOX) was used for its regeneration. The kinetics of all used enzymes was examined. Oxidation of lactol catalysed by AlDH was described by double-substrate Michaelis-Menten model with include substrate (NAD+) inhibition and with competitive inhibition by product (NADH). The kinetics of the same reaction catalysed by KRED was described by double-substrate Michaelis-Menten model with substrate (NADP+) inhibition and with competitive inhibition by product (NADPH). In the reaction of coenzyme regeneration, the kinetics of NOX was described by Michaelis-Menten model with competitive product (NADP+) inhibition. Beside kinetic measurements, the influence of the aldehydes on the stability of the AlDH, KRED and NOX was examined. Mathematical model of the process was developed and was validated by carrying out the reaction in a batch reactor. Experiments of lactol oxidation catalyzed by enzyme AlDH and KRED was carried out with coenzyme regeneration. As the substrates purified lactol as well the crude lactol, produced in the reaction of double aldol addition of acetaldehyde and chloroacetaldehyde catalyzed by 2-deoxyribose-5-phosphate aldolase (DERA) were used.

Cilj ovog rada je prezentirati različite matematičke modele iz područja kemije, fizike i biologije opisane običnim diferencijalnim jednadžbama. U prvom, uvodnom, poglavlju, upoznali smo se s osnovnim konceptima matematičkog modeliranja te svojstvima običnih diferencijalnih jednadžbi koje ćemo koristiti u nastavku. U drugom poglavlju, izvodom niza matematičkih modela promatrali smo mijenjanje veličine populacije. U trećem poglavlju smo koristeći zakon o djelovanju masa promatrali kinetiku enzima kroz tri različite reakcije: kompetitivna inhibicija, alosterička inhibicija i kooperativnost. U četvrtom poglavlju izveli smo razne modele bolesti populacije te iznijeli zaključke o uvjetima širenja zaraze. The purpose of this work is to present various mathematical models in the field of chemistry, physics and biology, all of them described by ordinary differential equations. In the first, introductory, chapter, we recalled the basic concepts of mathematical modeling and the properties of ordinary differential equations that we used in the sequel. In the second chapter, we have studied the population dynamics by formally deriving several mathematical models. In the third chapter, we have addressed the enzyme kinetics by employing the law of mass action via three different reactions: competitive inhibition, allosteric inhibition and cooperativity. In the fourth chapter, we have derived various models of population diseases and made conclusions regarding the conditions of spreading the disease.

U ovom radu ispitivana je kinetika reakcije redukcije acetaldehida uz biokatalizator ketoreduktazu koja za svoju aktivnost zahtijeva uporabu koenzima nikotinamid-adenin dinukleotid fosfata. Kinetika je mjerena metodom početnih brzina variranjem koncentracija supstrata (acetaldehida) i koenzima (NADPH) te su procijenjeni kinetički parametri Michaelis-Menteničine kinetike. Ispitivan je i utjecaj produkata na brzinu reakcije, te aktivnost enzima u povratnoj reakciji oksidacije etanola. Mjerenja su pokazala da produkt NADP+ inhibira reakciju, te da enzim ne pokazuje aktivnost u reakciji oksidacije etanola. Također je izmjerena stabilnost biokatalizatora pri različitim koncentracijama acetaldehida, te se pokazalo da povećanje koncentracije acetaldehida ima negativan utjecaj na stabilnost enzima. Na osnovu kinetičkih mjerenja i mjerenja stabilnosti biokatalizatora postavljen je matematički model redukcije acetaldehida u kotlastom reaktoru, te su napravljene simulacije procesa. In this paper the kinetics of acetaldehyde reduction catalysed by an ketoreductase was investigated. For its activity, ketoreductase demands the use of coenzyme nicotinamide-adenine dinucleotide phosphate. The kinetics was measured by determining the initial reaction rate by changing the concentrations of substrate (acetaldehyde) and coenzyme (NADPH). These data were used for the estimation of the kinetic parameters of Michaelis-Menten equation. The influence of products on the reaction rate, and the activity of enzyme in the reverse reaction, oxidation of ethanol, was also examined. Measurements have shown that the product NADP+ inhibits the reaction, and that the enzyme doesn't show activity in the reaction of ethanol oxidation. Stability of enzyme was measured in the presence of different concentrations of acetaldehyde, and it was shown that increase of acetaldehyde concentration have negative effect on the stability of the enzyme. Based on experimental data and the enzyme stability measurements, mathematical model was set for the reaction of reduction of acetaldehyde in the batch reactor, and simulations of the process have been done.

Statini su heterogena skupina lijekova koja se primjenjuje za snižavanje razine kolesterola u krvotoku čovjeka s ciljem prevencije kardiovaskularnih bolesti. Statini posjeduju 3,5-dihidroksi kiselinski bočni lanac s dva kiralna središta. Radi velikih zahtijeva kemijske i stereokemijske čistoće, njihova sinteza predstavlja izazov. U ovom radu provedena je biokatalitička oksidacija α-kloroaldehida (laktola) u aktivni kiselinski oblik bočnog lanca statina. Reakcija je katalizirana enzimom aldehid dehidrogenazom (AlDH) uz regeneraciju koenzima NAD+ enzimom NADH oksidazom (NOX). Ispitan je utjecaj različitih pH na aktivnost i stabilnost enzima AlDH i NOX. Kao optimalni uvjet za provedbu spomenute reakcije pokazala se vrijednost pH 8. Enzimi su u optimalnom mediju kinetički okarakterizirani u svim stupnjevima reakcije. Kinetika enzima AlDH u reakciji oksidacije laktola opisana je dvosupstratnom Michaelis-Menteničinom kinetikom s kompetitivnom inhibicijom supstratom (NAD+) i kompetitivnom inhibicijom produktom (NADH). Za kinetiku povratne reakcije pretpostavljeno je da se radi o kinetici drugog reda, a kako bi se procijenio kinetički parametar, provedena je oksidacija laktola bez regeneracije koenzima. Kinetika enzima NOX u reakciji oksidacije NADH opisana je Michaelis-Menteničinom kinetikom s kompetitivnom inhibicijom produktom (NAD+) te je nadograđena nekompetitivnom inhibicijom laktolom. Razvijen je matematički model procesa te je validiran provedbom reakcije u kotlastom reaktoru. Usporedno su provedena tri eksperimenta s različitim početnim koncentracijama koenzima. Proveden je i eksperiment u kojem je kao supstrat poslužio sirovi produkt (nepročišćeni laktol) dvostruke aldolne adicije acetaldehida i kloroacetaldehida katalizirane enzimom 2-deoksiriboza-5-fosfat aldolazom (DERA). Kako bi se mogla pratiti inaktivacija enzima tijekom provedbe eksperimenata, razvijeni su testovi za spektrofotometrijsko određivanje aktivnosti svakog pojedinog enzima. Kao potencijalno bolja metoda regeneracije koenzima, ispitan je postupak s lawsone kinonom umjesto enzima NOX. Statins are heterogeneous group of drugs used for lowering cholesterol level in human bloodstream preventing cardiovascular diseases. Structurally, they possess 3,5-dihydroxy acid side chain with two chiral centers. Synthesis of statine side chains is a challenge due to high demands for chemical and stereochemical purity. In this work a biocatalytical oxidation of α-chloroaldehyde (lactol) was carried out. Reaction is catalyzed by aldehyde dehydrogenase (AlDH) with coenzyme regeneration by NADH oxidase (NOX). The effect of various pH on activity and stability of both enzymes was examined. pH 8 was chosen as the optimal for the implementation of investigated reaction. Enzymes were kinetically characterized in all steps of the investigated reactions. Kinetics of AlDH in reaction of lactol oxidation was described by double-substrate Michaelis-Menten model with competitive inhibition by substrate (NAD+) and with competitive inhibition by product (NADH). Kinetics of the reverse reaction was assumed to be of the second order and to assess kinetic constant, lactol oxidation without coenzyme regeneration was performed. In reaction of coenzyme regeneration the kinetics of NOX was described by Michaelis-Menten model with competitive inhibition by product (NAD+) and was upgraded by non-competitive lactol inhibition. Mathematical model of the process was developed and was validated by carrying out the reaction in a batch reactor. Three parallel experiments with different initial coenzyme concentrations were conducted. Oxidation of lactol was also carried out with the crude product (lactol) using reaction mixture from double aldol addition of acetaldehyde and chloroacetaldehyde catalyzed by 2-deoxyribose-5-phosphate aldoase (DERA). In order to monitor enzyme inactivation during the experiments, tests for spectrophotometric AlDH and NOX activity determination were developed. As potential alternative for enzyme regeneration, procedure with lawsone quinone instead NOX was investigated.

Ketoreduktaze su enzimi koji sudjeluju u prijenosu elektrona, vodikovih ili kisikovih atoma te time kataliziraju redoks reakcije. U ovom radu provedeno je ispitivanje kinetike ketoreduktaze (KRED) u reakciji redukcije acetona, kao i ispitivanje kinetike u povratnoj reakciji, oksidaciji izopropanola. KRED za redukciju acetona zahtijeva prisustvo koenzima nikotin-amid-adenin-dinukleotid-fostata (NADPH) koji dovodi vodik do acetona reducirajući ga u izopropanol, prilikom čega NADPH prelazi u oksidirani oblik, NADP+. Eksperimentalni podaci obrađeni su u programskom paketu Scientist, a kinetički parametri procijenjeni su nelinearnom regresijom pomoću simpleks metode ili metode najmanjih kvadrata. Također, određena je stabilnost enzima u otopini acetona. Na temelju sheme reakcije i procijenjenih kinetičkih parametara postavljen je matematički model. Upotrebom modela provedena je simulacija redukcije acetona katalizirane ketoreduktazom u kotlastom reaktoru. Zaključeno je kako se reakcija redukcije acetona katalizirana ketoreduktazom može koristiti u procesu regeneracije koenzima NADP+ zbog postignute ravnotežne konverzije od 84,7 %. Ketoreductase is an enzyme involved in the transfer of electrons, hydrogen or oxygen atoms, thus catalyzing the redox reaction. In this paper, ketoreductase kinetics (KRED) was investigated in the reaction of acetone reduction as well as the kinetic in the reverse reaction, the isopropanol oxidation. KRED for acetone reduction requires the presence of nicotinamide-adenine-dinucleotide-phostate (NADPH) coenzyme, which transfers the hydrogen to acetone by converting it into isopropanol, whereby NADPH passes into an oxidized form, NADP+. Experimental data were processed in the Scientist program package, and kinetic parameters were estimated by nonlinear regression using the simplex method or the least squares method. Also, the stability of the enzyme in the acetone solution was determined. Based on reaction scheme and estimated kinetic parameters, a mathematical model was set. Using the model, the simulation of acetone reduction catalyzed by ketoreductase was done in the batch reactor. It was concluded that reaction of acetone reduction catalyzed by examine ketoreductase can be used in the NADP+ coenzyme regeneration process due to achieved 84,7 % equilibrium conversion.

Statini predstavljaju skupinu lijekova sa sposobnošću snižavanja kolesterola LDL (engl. Low Density Lipoprotein; LDL) i lipoproteina male gustoće inhibiranjem HMG-CoA (3-hidroksi-3-metilglutaril-koenzimA) reduktaza. Inhibicija navedenog enzima je vrlo važna u sprečavanju bolesti poput hiperkolesterolemije, koronarne bolesti, srčanih te kardiovaskularnih bolesti. U radu je provedena simulacija sinteze 6-kloro-3,5-dihidroksi heksanala, (laktola) koji je bitan prekursor u dobivanju statina. Sinteza je provedena u reakciji dvostruke aldolne adicije acetaldehida i kloroacetaldehida katalizirane enzimom 2-deoksiriboza-5-fosfat aldolazom (DERA). Određena je kinetika svih reakcija koje se odvijaju tijekom sinteze laktola katalizirane enzimom DERA te su kinetike opisane Michaelis-Menteničinim izrazima. Postavljen je matematički model sinteze laktola u kotlastom reaktoru, kotlastom reaktoru s dotokom i protočno kotlastom reaktoru. U model je također uključena deaktivacija enzima uzrokovana prisutnosti aldehida koje imaju negativan utjecaj na stabilnost enzima, te je opisana kinetikom drugog reda. Na temelju dobivenih rezultata simulacije, uspoređeni su različiti početni uvjeti sinteze laktola u različitim tipovima reaktora. Statins represents a group of medicines with the ability to lower cholesterol LDL and low density lipoproteins by inhibition HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme A), which is very important in the prevention of diseases such as hypercholesterolemia, coronary diseases, cardiac and cardiovascular diseases. In this study the simulation of the synthesis of an important precursor of statin, (3R, 5R)-6-chloro-3,5-dihydroxyhexanal (lactone) was carried out. The synthesis was performed in the reaction of double aldol addition of acetaldehyde and chloroacetaldehyde catalyzed by the enzyme 2-deoxyribose-5-phosphate aldolase (DERA-e). The kinetics of all reactions that occurs during lactol synthesis catalyzed by DERA has been determined and kinetics was described by Michaelis-Menten equations. A mathematical model of lactol synthesis in batch reactor, fed-batch reactor and continuous tank flow reactor was established. The model also included the enzyme deactivation caused by the presence of various aldehydes that have a negative impact on the enzyme stability and was described by the second order kinetics. Based on the results of the simulation, the different initial conditions of lactol synthesis in different reactor mode were compared.

Statini su heterogena skupina lijekova koja se koristi pri snižavanju kolesterola te sprečavanju pojave kardiovaskularnih bolesti. Strukturno svi statini posjeduju 3,5-dihidroksi kiselinski bočni lanac s dva kiralna centra kojeg je teško sintetizirati zbog potrebe za visokom kemijskom i stereokemijskom čistoćom. U radu je provedena sinteza (3R,5R)-6-kloro-3,5-dihidroksiheksanala (laktola), prekursora za dobivanje statina. Sinteza se provela dvostrukom aldolnom adicijom acetaldehida i kloroacetaldehida kataliziranom enzimom 2-deoksiriboza-5-fosfat aldolaza (DERA). Ispitani su utjecaji različitih pufera i pH vrijednosti na stabilnost i aktivnost enzima DERA. Kao najoptimalniji uvjeti (pufer i pH) pokazali su se 100 mM fosfatni pufer i pH 6 pri kojim je enzim DERA kinetički okarakteriziran u svim stupnjevima reakcije. U reakciji acetaldehida i kloroacetaldehida te u reakciji acetaldehida i 4-kloro-3-hidroksibutanala kinetika je opisana dvosupstratnom Michaelis-Menteničinom jednadžbom dok je kinetika enzima u reakciji trimerizacije acetaldehida opisana kinetikom prvog reda, te je postavljen matematički model procesa u različitim tipovima reaktora. Ispitana je deaktivacija enzima u prisutnosti različitih koncentracija acetaldehida, kloroacetaldehida i međuprodukta, 4-kloro-3-hidroksibutanala, te je opisana kinetikom drugog reda. Sinteza laktola i validacija modela je provedena u tri različita tipa reaktora (kotlasti reaktor, ponovljivi kotlasti reaktor i kotlasti reaktor s dotokom) kako bi se dobio uvid i pronašlo najbolje rješenje za prevladavanje nedostataka stabilnosti korištenog enzima. Statins are a heterogeneous group of drugs used to reduce cholesterol and prevent the phenomenon of cardiovascular disease. Structurally, all statins possess a 3,5-dihydroxy acid side chain with two chiral centers which is difficult to synthesize due to the need for high chemical and stereochemical purity. In this study the synthesis of (3R,5R)-6-chloro-3,5-dihydroxyhexanal (lactol), a precursor for the preparation of statin was investigated. The synthesis was performed in the reaction of double aldol addition of acetaldehyde and chloroacetaldehyde catalyzed by the enzyme 2-deoxyribose-5-phosphate aldolase (DERA). The influence of different buffers and pH values on the stability and activity of the DERA enzyme was tested. 100 mM phosphate buffer and pH 6 were chosen as the optimal conditions (buffer and pH). Enzyme DERA was kinetically characterized in all steps of the investigated reactions. In the reaction of acetaldehyde and chloroacetaldehyde and in reaction of acetaldehyde and 4-chloro-3-hydroxybutane kinetics was described by the two-substrate Michaelis - Menten equation, while the kinetics of the enzyme in the reaction of acetaldehyde trimerization is described by the first order kinetics. Mathematical model of the process was proposed in different types of reactor. Enzyme deactivation was tested in the presence of different concentrations of acetaldehyde, chloroacetaldehyde and intermediate, 4-chloro-3-hydroxybutanal, and was described by second order kinetics. The lactol synthesis and validation of the model were carried out in three different types of reactors to gain insight and find the best solution to overcome the stability deficiencies of used enzyme.

Šećerna bolest je metabolički poremećaj kojeg karakterizira stanje kronične hiperglikemije i poremećeni metabolizam ugljikohidrata, masti i proteina zbog oštećene sekrecije inzulina i/ili poremećaja u signalnim putevima. U održavanju odgovarajuće koncentracije glukoze u krvi ključnu ulogu ima jetra. Metabolizam glukoze u jetri reguliraju hormoni koji utječu na enzimsku aktivnost ili sintezu samog enzima. Ovisno o potrebama metabolizma izmijenjuju se dva procesa, glikoliza i glukoneogeneza. Kako bi se koncentracija glukoze održala unutar referentnog intervala bitna je regulacija ključnih enzima oba procesa. U šećernoj bolesti dolazi do povećane glukoneogeneze i glikogenolize u stanicama jetre. S druge strane smanjene su aktivnosti enzima glikolize i glikogeneze. Cilj ovog istraživanja je ispitati stupanj glikolize u tumorskim HepG2 stanicama u hiperglikemijskim uvjetima. HepG2 stanice tretirali smo s četiri otopine različitih koncentracija glukoze (5 mM, 20 mM, 30 mM i 50 mM) te su tako tretirane stanice predstavljale model hepatocita u šećernoj bolesti. Stupanj glikolize odredili smo na temelju mjerenja katalitičke aktivnosti glikolitičkog enzima piruvat kinaze. Piruvat kinaza ima ključnu ulogu u zadnjem koraku glikolize, prevođenju fosfoenolpiruvata u piruvat. Aktivnost jetrenog izoenzima regulirana je alosteričkim efektorima i fosforilacijom. Katalitičku aktivnost piruvat kinaze (PK) izračunali smo iz spektrofotometrijskog mjerenja apsorbancije reduciranog koenzima. Podaci su statistički obrađeni u komercijalnom statističkom programu GraphPad Prism 6.01 i Microsoft Office Excel 2016. Za obradu rezultata korišteni su one-way ANOVA i Sidakov test. Rezultati istraživanja pokazuju statistički značajan pad aktivnosti piruvat kinaze kako se povećavala koncentracija glukoze u stanicama. Katalitička aktivnost enzima bila je najniža u uvjetima 30 mM glukoze. Kod koncentracije 50 mM glukoze nije vidljiv statistički značajni pad u odnosu na kontrolu jer je došlo do zasićenja aktivnih mjesta na enzimu. Rezultati potvrđuju prethodne hipoteze da je stupanj glikolize manji kod osoba s uznapredovalim stadijem šećerne bolesti kod kojih je prisutna veća koncentracija glukoze u krvi (20 mM i 30 mM). Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia and disturbed metabolism of carbohydrates, fats and proteins that occurs due damaged insulin secretion or disorder in insulin signal pathways. Liver has the essential role in maintenance adequate glucose concentration in blood. Glucose metabolism in liver is regulated by hormones which effect enzyme activity or enzyme synthesis. Depending on the metabolic needs, glycolysis and gluconeogenesis are exchanged. Regulating the essential enzymes of these processes is important to maintain glucose concentration within reference range. In diabetes mellitus, gluconeogenesis and glycogenolysis in liver cells are increased. On the other hand, activity of glycolytic and glycogenesis enzymes is decreased. The aim of this study was to examine the rate of glycolysis in tumor HepG2 cells in hyperglycemic conditions. HepG2 cells were treated with four different glucose concentrations (5 mM, 20 mM, 30 mM and 50 mM) and such treated cells represented a model of hepatocytes in diabetes mellitus. The rate of glycolysis was determined by measurements of pyruvate kinase catalytic activity. Pyruvate kinase has the essential part in the last step in glycolysis, conversion phosphoenolpyruvate in pyruvate. Liver isoenzyme activity is regulated by allosteric effectors and phosphorylation. We calculated the pyruvate kinase activity (PK) from the spectrophotometric measurements of reduced coenzyme. The results were statistically analyzed in the statistical program GraphPad Prism 6.01 and Microsoft Office Excel 2016. For result analysis, we used one-way ANOVA and Sidak test. Research results show statistically significant drop of pyruvate kinase activity due to increased glucose concentration in cells. Catalytic enzyme activity was the lowest in 30 mM glucose conditions. In 50 mM glucose concentration, there was no statistically significant drop regards to control because saturation od active sites on the enzyme was occurred. Results confirm previous hypothesis that the rate of glycolysis is lower in people with advanced stage of diabetes mellitus with higher blood glucose concentrations (20 mM and 30 mM).

Enzimi su katalizatori u biološkim sustavima. Ubrzavaju reakcije snižavanjem energije aktivacije. Gotovo svi poznati enzimi su proteini. Najvažniji dio enzima je aktivno mjesto u kojem se odvija specifiĉna reakcija. Butirilkolinesteraza (BuChE) pripada skupini kolinesteraza odnosno estereaza. Esteraze spadaju u grupu hidrolaza. BuChE inaktivira neurotransmiter acetilkolin (ACh) i kao takva je postala terapeutski cilj u poodmaklim fazama Alzheimerove bolesti. Kao izvor enzima korištena je butirilkolinesteraza (BuChE) iz seruma konja, a kao supstrat enzima korišten je butiriltiokolin jodid (BuTChI). Kao inhibitor enzima korišten je α-terpinolen. Iz Lineweaver-Burk, Hanes-Wolf i Eisenthal – Cornish-Bowden prikaza te izraĉunatih postotaka inhibicije zakljuĉeno je da α-terpinolen pokazuje sposobnost inhibicije enzima BuChE. Kako se povećava koncentracija α-terpinolena povećava se i postotak inhibicije. Također je zakljuĉeno da se radi o inhibiciji miješanog tipa te su određeni odgovarajući kinetiĉki parametri KM, Vmax, KMapp, Vmaxapp, KI i KI'. Enzymes are catalysts in biological systems. Accelerate reactions by lowering the activation energy. Nearly all known enzymes are proteins. The most important part of the enzyme is their active site where reactions take place. Butyrylcholinesterase (BuChE) belongs to a class of cholinesterase or esterase. Esterases belong to the group of hydrolases. BuChE inactivates the neurotransmitter acetylcholine (ACh) and as such has become a therapeutic target in later stages of Alzheimer's disease. Butyrylcholinesterase (BuChE) enzyme was obtained from the horse serum, and as a substrate of the enzyme was used butyrylthiocholine iodide (BuTChI). The enzyme used was α-terpinolene. From Lineweaver-Burk, Hanes-Wolf and Eisenthal - Cornish-Bowden display and calculated the percent inhibition was concluded that α-terpinolene shows the ability to inhibit the enzyme BuChE. α-Terpinolene inhibits BuChE in dose dependent manner. It was also concluded that it is a mixed type inhibition and certain corresponding kinetic parameters KM, Vmax, KMapp, Vmaxapp, KI and KI'.

Enzimi su biokatalizatori. Oni kataliziraju reakcije tako što stabiliziraju prijelazno stanje. Visoko su specifični po reakcijama koje kataliziraju kao i po supstratima, najčešće kataliziraju samo jednu reakciju ili nekoliko srodnih reakcija. Najvažniji dio enzima je aktivno mjesto u kojem se odvija specifična reakcija. Acetilkolinesteraza (AChE) je enzim iz skupine hidrolaza. Katalizira reakciju hidrolize neuroprijenosnika acetilkolina na acetat i kolin. Kao izvor enzima korištena je AChE iz elektrofora električne jegulje (Tip V-S), a kao supstrat enzima korišten je acetiltiokolin-jodid (ATChI) dok je α-terpinolen ispitivan na inhibiciju AChE. Inhibicija acetilkolinesteraze ima ključnu ulogu u ublažavanju simptoma kod Alzheimerove demencije. Cilj ovoga rada bio je ispitati sposobnost inhibicije enzima acetilkolinesteraze korištenjem α-terpinolena kao inhibitora te odrediti vrstu inhibicije. Iz Lineweaver-Burk, Eadie-Hofstee, Hanes-Wolf i Eisenthal – Cornish-Bowden prikaza te izračunatih postotaka inhibicije zaključeno je da α-terpinolen pokazuje sposobnost inhibicije enzima AChE. Povećanjem koncentracije α-terpinolena povećava se i postotak inhibicije, a najveća vrijednost inhibicije je postignuta pri koncentraciji α-terpinolena od 0,0227 mg mL-1 i iznosi 91,8 %. Također je zaključeno da se radi o inhibiciji miješanog tipa te su određeni odgovarajući kinetički parametri KM, Vmax, KMapp, Vmaxapp, KI i K'I. Enzymes are biocatalysts. They catalyse reactions by stabilizing the transition state and are highly specific for reactions that they catalyse as well as their substrates. Enzymes usually catalyse only one or small number of similar reactions. The most important part of the enzyme is their active site where reactions take place. Acetylcholinesterase (AChE) is an enzyme that belongs to the hydrolases group and catalyses the hydrolysis of the acetylcholine neurotransmitter to acetate and choline. In this study AChE from the electrophores of electric eel (type V-S) was used as a source of the enzyme and acetylthiocholine iodide (ATChI) was used as a substrate while α-terpinolene was tested for AChE inhibitory activity. Inhibition of acetylcholinesterase has a crucial role in the treatment of Alzheimer's dementia. The aim of this study was to examine the ability of α-terpinolene to inhibit AChE and to determine the type of inhibition. After reviewing the results using Lineweaver-Burk, Eadie-Hofstee, Hanes-Wolf and Eisenthal – Cornish-Bowden plots as well as calculating the inhibition percentage it was concluded that α-terpinolene shows ability to inhibit the AChE in concentration dependant manner. Maximum percentage of AChE inhibition, 91,8 %, was achieved at α-terpinolene concentration of 0,0227 mg mL-1. It was, also, concluded that α-terpinolene exhibits a mixed type inhibition and appropriate kinetic parameters were determined i.e. KM, Vmax, KMapp, Vmaxapp, KI i K'I.

Statini su heterogena skupina lijekova koji se primjenjuju u prevenciji i liječenju bolesti kardiovaskularnog sustava. U radu je provedena sinteza (3R,5R)-6-kloro-3,5-dihidroksi heksanala (laktona), važnog prekursora u dobivanju statina. Sinteza je provedena u reakciji aldolne kondenzacije kloroacetaldehida i acetaldehida katalizirane enzimom 2-deoksiriboza-5-fosfat aldolazom (DERA). Ispitana je stabilnost i aktivnost enzima DERA-e pri različitim temperaturama i puferima pri različitim pH vrijednostima. Kao optimalni uvjeti za provedbu reakcije aldolne kondenzacije katalizirane enzimom DERA pokazali su se temperatura od 25°C i 0,1 M TEA-HCl pufer pH 7. Ispitana je deaktivacija enzima u prisutnosti različitih koncentracija acetaldehida i kloroacetaldehida te je opisana kinetikom drugog reda. Utvrđeno je da konstanta deaktivacije eksponencijalno raste s koncentracijom acetaldehida i kloroacetaldehida prisutnih u sustavu. Enzim je kinetički karakteriziran te je kinetika opisana dvosupstratnom Michaelis-Menteničinom jednadžbom. Postavljen je matematički model sinteze laktona te je provedena ocjena valjanosti u kotlastom reaktoru, ponovljivom kotlastom reaktoru te kotlastom reaktoru s dotokom. Statins are a heterogeneous group of medications used in prevention and treatment of cardiovascular diseases. In this study the synthesis of an important precursor of statin, (3R, 5R)-6-chloro-3,5-dihydroxyhexanal (lactone) was carried out. The synthesis was performed in the reaction of aldol condensation of chloroacetaldehyde and acetaldehyde catalyzed by the enzyme 2-deoxyribose-5-phosphate aldolase (DERA-e). In order to find optimal conditions for lactone synthesis in the reaction of aldol condensation, stability and activity of the enzyme were tested at different temperatures and in different buffer solutions at different pH values. The optimal conditions were found to be: 25°C and 0,1 M TEA-HCl buffer pH 7. Enzyme deactivation in a presence of different concentrations of acetaldehyde and chloroacetaldehyde was investigated. Deactivation was described with a second-order kinetic model. It was observed that a deactivation constant increases exponentially with the concentration of acetaldehyde and chloroacetaldehyde. The enzyme was kinetically characterized and kinetics was described by two-substrate Michaelis-Menten equation. Mathematical model of lactone synthesis was proposed and was validated in a batch, repetitive batch and fed-batch reactor.