| Abstract | Statini su učestalo korišteni lijekovi za sniženje kolesterola niske gustoće u krvi. Zajednička karakteristika svim statinima je (3R, 5R/S)-dihidroksiheksanoatski bočni lanac koji sadrži dva kiralna centra, čija sinteza predstavlja izazov za konvencionalne kemijske metode. Iz tog razloga, sve se više razvijaju alternativne metode sinteze korištenjem biokatalizatora, koji omogućuju dobivanje čistog stereoizomera korištenjem jeftinih akiralnih supstrata.
Glavni nedostatak ove metode je niska stabilnost enzima, te sklonost deaktivaciji pri višim koncentracijama supstrata. Iz tog razloga je u ovom doktorskom radu detaljno ispitana kovalentna imobilizacija enzima 2-deoksi-D-riboza 5-fosfat aldolaza (DERA024) na različite tipove nosioca u reakciji dvostruke aldolne adicije acetaldehida i kloroacetaldehida,
s obzirom da imobilizacija može značajno povećati stabilnost enzima uz zadržavanje njegove aktivnosti. U tu svrhu prvo je izvršena sinteza dvije vrste nosioca, mezoporozne silike (engl. mesostructured celluar foam, MCF) i magnetskih nanočestica (engl. magnetic nanoparticles, MNP), koji su idealni kandidati za ovaj tip imobilizacije s obzirom na svoju mehaničku i kemijsku čvrstoću, biokompatabilnost i inertnost. Nakon izvršene sinteze napravljena je detaljna karakterizacija oba nosioca kako bi se utvrdile karakteristike materijala koje uključuju morfologiju, veličinu čestica i promjer pora. Kako bi kovalentna imobilizacija enzima na sintetizirane nosioce bila moguća, potrebno je na nosioce vezati reaktivne skupine.
U tu svrhu ispitane su različite kombinacije i koncentracije funkcionalizacijskih
(3-aminopropiltrietoksilan (APTES), (3-metilaminopropil)trimetoksilan (APTMS) i
(3-glicidiloksipropil)trimetoksilan (GPTMS)) i aktivacijskih (glutaraldehid, benzokinon, anhidrid jantarne kiseline) agensa pri čemu je praćen njihov utjecaj na procesne pokazatelje procesa imobilizacije, a to su uspješnost imobilizacije, očuvanost aktivnosti enzima te procesna stabilnost enzima. Nakon definiranja najučinkovitije kombinacije ispitivanih agensa, ispitan je utjecaj temperature i pH na proces imobilizacije. Kotlasti reaktor je u praksi jedna od najčešće korištenih izvedbi reaktora u reakcijama aldolne adicije ovog tipa. Stoga je stabilnost MCF/MNP-imobiliziranog enzima ispitana u kotlastom reaktoru kroz više ciklusa, a dobiveni rezultati uspoređeni su s rezultatima praćenja stabilnosti slobodnog enzima.
S obzirom da je mezoporozna silika porozna te da to svojstvo može utjecati na procesne pokazatelje imobilizacije, a posljedično na karakteristike enzima i samu reakciju, dodatno je ispitan utjecaj veličine pora. Na ovaj način dobiveni su važni zaključci o utjecaju poroznosti na ovdje ispitivanu reakciju. Za izradu matematičkog modela procesa s obje vrste nosioca, provedena su kinetička mjerenja pri čemu je promatran utjecaj oba supstrata i međuprodukta na specifičnu aktivnost enzima koristeći metodu početnih brzina. Iz dobivenih podataka procijenjeni su parametri Michaelis-Menteničine kinetike primjenom nelinearne regresijske analize dostupne u programskom paketu Scientist (MicroMath). Korištenjem procijenjenih kinetičkih parametara te bilančnih jednadžbi, razvijen je matematički model procesa s oba nosioca. Dobiveni rezultati uspoređeni su s onima slobodnog enzima kako bi se dobio detaljniji uvid u promjene i ponašanje procesa s imobiliziranim enzimom. Ocjena valjanosti razvijenog matematičkog modela provedena je na nezavisnom setu eksperimentalnih podataka dobivenih provedbom procesa u kotlastom reaktoru (primjena MCF i MNP), te u kotlastom reaktoru s dotokom (primjena MNP). U zadnjem dijelu, u svrhu intenzifikacije procesa, reakcija sinteze prekursora statina provedena je u cijevnom reaktoru primjenom imobiliziranog enzima. U slučaju MCF ispitan je cijevni reaktor s nasutim slojem katalizatora, dok je kod MNP ispitan cijevni reaktor s fluidiziranim slojem katalizatora u promjenjivom magnetskom polju. Tijekom procesa praćen je utjecaj različitih protoka i vremena zadržavanja na procesne pokazatelje, konverziju, produktivnost, selektivnost i iskorištenje. Na osnovu dobivenih rezultata izvučeni su zaključci o učinkovitosti primjene oba nosioca u kontinuiranim reaktorskim sustavima. Na temelju rezultata dobivenih u ovom istraživanju, može se zaključiti da je kovalentna imobilizacija vrlo učinkovita metoda za stabilizaciju enzima DERA024 korištene u reakciji dvostruke aldolne adicije acetaldehida i kloroacetaldehida s ciljem proizvodnje prekursora statina. Osim toga, postignuta su značajna poboljšanja aktivnosti i stabilnosti enzima za oba ispitana nosioca. Ovdje razvijeni i ocjenjeni matematički modeli predstavljaju dobre temelje za daljnju optimizaciju i primjenu ovakvog tipa imobilizacije za poboljšanje procesa enzimatske sinteze prekursora statina. |
| Abstract (english) | Statins are the most commonly used drugs for lowering low-density cholesterol levels in the blood. Common to all statins is the (3R, 5R/S)-dihydroxyhexanoate side chain, which contains two chiral centers, the synthesis od which is a challenge for conventional chemical synthesis methods. For this reason, alternative synthesis methods using biocatalysts are increasingly being developed, which enable the production of pure stereoisomers with usage of inexpensive achiral substrates. The main drawback of this method is the low stability of the enzymes and their tendency to deactivate at higher substrate concentrations. For this reason, the covalent immobilization of the enzyme 2-deoxy-D-ribose-5-phosphate aldolase (DERA024) on different types of supports in the reaction of double aldol addition of acetaldehyde and chloroacetaldehyde was investigated in detail in this thesis, with assumption that immobilization can significantly increase the stability of the enzyme while maintaining its activity. The synthesis of two types of carriers, mesoporous silica (MCF) and magnetic nanoparticles (MNP), which are ideal candidates for this type of immobilization due to their mechanical and chemical robustness, biocompatibility and inertness, was first carried out. A detailed characterization of the two carriers was performed with several methods to confirm the obtained material properties such as morphology, particle size and pore size.
In order to achieve covalent immobilization, it is necessary to bind reactive groups to the carriers that are able to form a covalent bond with the enzyme. For this purpose, different combinations and concentrations of functionalizing ((3-aminopropyl)triethoxysilane (APTES), ((3-aminopropyl)trimethoxysilane (APTMS), ((3-Glycidyloxypropyl)trimethoxysilane (GPTMS)) and activating (benzoquinone, glutaraldehyde, succinic anhydride) agents were tested for both carriers to determine their effects on the process indicators of immobilization, which include yield, retained activity and stability. Once the ideal combination of tested agents was found, the influence of temperature and pH on the immobilization process itself was investigated. Since a batch reactor is one of the most commonly used reactor design for reactions of this type, the stability of the MCF/MNP-immobilized enzyme in a batch reactor was tested over several cycles and compared with the stability of the free enzyme. Due to the porosity of mesoporous silica and its ability to influence the properties of the enzyme and the reaction, the effect of pore size was further investigated and relevant conclusions were drawn on the effects of porosity. To develop a mathematical model of the reaction with both types of support, kinetic measurements were performed in which the effects of the substrates and intermediates on the specific activity of the enzyme were observed using the initial rate method. From the data obtained, the parameters of Michaelis-Menten kinetics were determined using the nonlinear regression analysis available in the Scientist software package (MicroMath). Using the obtained parameters and reactor equations, a mathematical model of the process with both carriers was developed. The calculated parameters and the developed models were compared with those of the free enzyme to gain a more detailed insight into the changes caused by immobilization and potential process improvements.
The developed models were experimentally validated in a batch reactor for both carriers and in a fed-batch reactor for magnetic nanoparticles. In the last part of the research work,
the effects of different flow rates and residence times on process indicators of conversion, productivity, selectivity and yield were investigated in a continous tubular reactor. In the case of mesoporous silica, a tubular reactor with a catalyst packed bed was tested, while for magnetic nanoparticles a tubular reactor with a catalyst fluidized bed in a variable magnetic field was investigated. Based on these results, conclusions were drawn about the efficiency of using both carriers in continuous reactor systems. All results of this investigation suggest that covalent immobilization is a very effective choice for the stabilization of DERA024 enzyme in the double aldol addition reaction of acetaldehyde and chloroacetaldehyde for the production of statin precursors, and that significant improvements of enzyme activity and stability were achieved for both tested carriers. The mathematical models developed and validated here represent an important basis for the further optimization and application of this type of immobilization to improve the enzymatic synthesis process of statin precursors. |
