259 results on '"Findrik Blažević, Zvjezdana"'
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2. Impact of organic solvents on the catalytic performance of halohydrin dehalogenase
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Milčić, Nevena, Švaco, Petra, Sudar, Martina, Tang, Lixia, Findrik Blažević, Zvjezdana, and Majerić Elenkov, Maja
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- 2023
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Catalog
3. Optimization of Biocatalytic Rhododendrol Production from Biogenic Rhododendrol Glycosides.
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Leaković, Emerik, Siems, Karsten, Feussi Tala, Michel, Habazin, Antonia, Findrik Blažević, Zvjezdana, and Vrsalović Presečki, Ana
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- 2024
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4. Methanogenesis—General Principles and Application in Wastewater Remediation.
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Marić, Ana-Katarina, Sudar, Martina, Findrik Blažević, Zvjezdana, and Vuković Domanovac, Marija
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INDUSTRIAL wastes ,WASTEWATER treatment ,ANAEROBIC reactors ,RENEWABLE natural gas ,BIOGAS production ,METHANOGENS - Abstract
The first discovery of methanogens led to the formation of a new domain of life known as Archaea. The Archaea domain exhibits properties vastly different from previously known Bacteria and Eucarya domains. However, for a certain multi-step process, a syntrophic relationship between organisms from all domains is needed. This process is called methanogenesis and is defined as the biological production of methane. Different methanogenic pathways prevail depending on substrate availability and the employed order of methanogenic Archaea. Most methanogens reduce carbon dioxide to methane with hydrogen through a hydrogenotrophic pathway. For hydrogen activation, a group of enzymes called hydrogenases is required. Regardless of the methanogenic pathway, electrons are carried between microorganisms by hydrogen. Naturally occurring processes, such as methanogenesis, can be engineered for industrial use. With the growth and emergence of new industries, the amount of produced industrial waste is an ever-growing environmental problem. For successful wastewater remediation, a syntrophic correlation between various microorganisms is needed. The composition of microorganisms depends on wastewater type, organic loading rates, anaerobic reactor design, pH, and temperature. The last step of anaerobic wastewater treatment is production of biomethane by methanogenesis, which is thought to be a cost-effective means of energy production for this renewable biogas. [ABSTRACT FROM AUTHOR] more...
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- 2024
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5. Biokataliza u doba zelene revolucije
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Glavinić, Laura, primary, Milčić, Nevena, additional, Findrik Blažević, Zvjezdana, additional, and Sudar, Martina, additional
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- 2024
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6. Multi-reaction kinetic modeling for the peroxidase–aldolase cascade synthesis of a D-fagomine precursor
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Masdeu, Gerard, Findrik Blažević, Zvjezdana, Kralj, Slavko, Makovec, Darko, López-Santín, Josep, and Álvaro, Gregorio
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- 2021
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7. An innovative route for the production of atorvastatin side-chain precursor by DERA-catalysed double aldol addition
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Švarc, Anera, Fekete, Melinda, Hernandez, Karel, Clapés, Pere, Findrik Blažević, Zvjezdana, Szekrenyi, Anna, Skendrović, Dino, Vasić-Rački, Đurđa, Charnock, Simon J., and Presečki, Ana Vrsalović
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- 2021
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8. A multi-enzyme strategy for the production of a highly valuable lactonized statin side-chain precursor
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Švarc, Anera, Findrik Blažević, Zvjezdana, Vasić-Rački, Đurđa, Charnock, Simon J., and Vrsalović Presečki, Ana
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- 2020
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9. HHDH-Catalyzed Synthesis of Enantioenriched Fluorinated β-Hydroxy Nitrile─Process Advances through a Reaction Engineering Approach
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Milčić, Nevena, primary, Sudar, Martina, additional, Marić, Ana-Katarina, additional, Kos, Krešimir, additional, Majerić Elenkov, Maja, additional, and Findrik Blažević, Zvjezdana, additional
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- 2024
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10. Biokataliza u doba zelene revolucije
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Glavinić, Laura, Milčić, Nevena, Findrik Blažević, Zvjezdana, Sudar, Martina, Glavinić, Laura, Milčić, Nevena, Findrik Blažević, Zvjezdana, and Sudar, Martina
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U dobu zelene revolucije sve više raste svijest o potrebi razvoja održivijih alternativa tradicionalnim industrijskim procesima. Gotovo svi industrijski procesi upotrebljavaju katalizatore koji su nerazgradivi i mogu biti štetni za okoliš. S druge strane, biokatalizatori su enzimi – molekule evoluirale u fiziološkom okruženju koje su u potpunosti biorazgradive, a u blagim reakcijskim uvjetima fiziološkog pH, temperature i tlaka okoline pokazuju najveću katalitičku aktivnost i učinkovitost. Biokatalizu ljudi primjenjuju još od starog vijeka u proizvodnji hrane poput sira, kiselog tijesta, piva, vina i octa bez znanja o pozadini procesa koji se odvija, a u posljednjih nekoliko desetljeća porasla je i njezina primjena u industriji, posebice u proizvodnji lijekova i kemikalija. Prepoznate su sve prednosti koje imaju biokatalizatori, poput njihove velike selektivnosti, velike specifičnosti prema supstratu i biorazgradivosti, te postaje sve jasnije da biokataliza može pomoći ispunjenju ciljeva održivog razvoja i implementaciji zelene kemije u industrijske procese s maksimalnim iskorištenjem resursa uz minimalno stvaranje otpada. Iako biokataliza ispunjava gotovo sva načela zelene kemije i potencijal biokatalizatora eksponencijalno raste razvojem inovacija i tehnološkog napretka, posebno u područjima biotehnologije i molekularne biologije, sama prisutnost biokatalizatora u kemijskom procesu ne podrazumijeva održivost procesa. Ekološku prihvatljivost, ali i ekonomsku isplativost, procesa je potrebno dokazati što ranije tijekom njegova razvoja praćenjem raznih procesnih pokazatelja kako bi se uštedjeli i vrijeme i novac., In the era of the Green Revolution, awareness of the necessity to develop more sustainable alternatives to traditional industrial processes is growing. Nearly all industrial processes utilise catalysts that are nondegradable and potentially harmful to the environment. On the other hand, biocatalysts are enzymes – molecules evolved in a physiological environment – that are entirely biodegradable. Under mild reaction conditions such as physiological pH, room temperature, and pressure, they exhibit their highest catalytic activity and efficiency. Biocatalysis has been utilised since ancient times in the production of food items such as cheese, sourdough, beer, wine, and vinegar, without the knowledge of the background of the process. Over the last few decades, its industrial application has also increased, especially in the production of medicines and chemicals. All the advantages of biocatalysts, such as their high selectivity, high specificity toward substrates, and biodegradability, have been acknowledged. It is becoming increasingly evident that biocatalysis represents one of the key means to accomplish Sustainable Development Goals and to implement green chemistry in industrial processes, maximising the use of resources while minimising waste generation. Even though biocatalysis aligns with almost all green chemistry principles, and the potential of biocatalysts is experiencing exponential growth through the evolution of innovations and technological advancements, the mere presence of a biocatalyst in a chemical process does not imply sustainability of the process. The environmental acceptability, as well as the economic viability of the process need to be demonstrated as early as possible in its development by monitoring various metrics to save both time and money. more...
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- 2024
11. Model-based optimization of the enzymatic aldol addition of propanal to formaldehyde: A first step towards enzymatic synthesis of 3-hydroxybutyric acid
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Česnik, Morana, Sudar, Martina, Roldan, Raquel, Hernandez, Karel, Parella, Teodor, Clapés, Pere, Charnock, Simon, Vasić-Rački, Đurđa, and Findrik Blažević, Zvjezdana
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- 2019
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12. Discovery of Plastics-degrading Enzymes
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Çakar, Mehmet Mervan, primary, Vuković Domanovac, Marija, additional, and Findrik Blažević, Zvjezdana, additional
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- 2023
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13. Discovery of Plastics-degrading Enzymes
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Mervan Çakar, Mehmet, Vuković Domanovac, Marija, Findrik Blažević, Zvjezdana, Mervan Çakar, Mehmet, Vuković Domanovac, Marija, and Findrik Blažević, Zvjezdana
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Plastics are highly advanced materials that have a vast array of applications and are produced globally in an approximate amount of 350 to 400 million tons every year. Nevertheless, there are serious concerns about plastic waste and pollution as a result of the misuse and lack of control of their use in industries, including packaging, transportation, manufacturing, and agriculture. Approximately 1,000 years are required for plastic bags to decompose efficiently. Additionally, CO2 and dioxins are released into the atmosphere by burning plastics, and they contribute to global warming. The Earth’s environment is overwhelmed with waste, mostly from poor recycling practices and low circular usage, resulting in millions of tons of waste generated annually. To combat this, new technologies for recycling post-consumer plastics are desperately needed to decrease plastic waste and improve the environment, while also finding ways to utilise these materials. Due to the inadequate disposal methods currently available for plastic waste, there has been increased interest in the use of microorganisms and enzymes designed for the biodegradation of non-degradable synthetic polymers via biocatalytic depolymerisation indicating that plastics treatment and recycling can be more efficient and sustainable., Plastika je daleko najnapredniji materijal kad je riječ o primjeni i svojstvima, a procjenjuje se da se svake godine globalno proizvede 350 do 400 milijuna tona. Ona je postala ozbiljan problem s obzirom na odlaganje plastičnog otpada i onečišćenje, zbog njezine nekontrolirane upotrebe u različite svrhe tijekom posljednjih desetljeća, kao što su pakiranje, transport, industrija i poljoprivreda. Za učinkovitu razgradnju plastičnih vrećica potrebno je otprilike 1000 godina. Osim toga, izgaranjem plastike u atmosferu se ispuštaju CO2 i dioksini koji doprinose globalnom zatopljenju. Zemaljski kopneni ili morski okoliš akumulira milijune tona otpada svake godine zbog lošeg recikliranja i niske kružne upotrebe. Inovativne tehnologije za recikliranje otpadne plastike prijeko su potrebne za smanjenje plastičnog otpada i postizanje ciljeva kvalitete okoliša uz valorizaciju potrošne plastike. Zbog trenutačno neadekvatnih metoda zbrinjavanja plastičnog otpada povećan je fokus na upotrebu mikroorganizama i enzima dizajniranih za biorazgradnju nerazgradivih sintetičkih polimera putem biokatalitičke depolimerizacije, što ukazuje na to da obrada plastike i recikliranje mogu biti učinkovitiji i održivi. more...
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- 2023
14. Synthesis of valuable building block with immobilized biocatalyst in rotating bed reactor
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Kos, Krešimir, Milčić, Nevena, Majerić Elenkov, Maja, Findrik Blažević, Zvjezdana, and Rogošić, Marko
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Halohydrin dehalogenase ,Fluorinated building blocks ,Immobilized biocatalyst ,Rotating bed reactor - Abstract
Halohydrin dehalogenases (HHDHs) are industrially important bacterial enzymes that belong to the group of lyases. On account on their activity, stability, β-regioselectivity and (R)- enantioselectivity, HHDHs can be used in the synthesis of optically active epoxides and β- substituted alcohols. Industrial applicability of biocatalysts increases with their stability and reusability, both of which can be improved with the use of different immobilization methods. Some of the most common methods of enzyme immobilization include covalent binding, adsorption, ionic binding, encapsulation, and cross-linking. Apart from enzymes, it is also possible to immobilize whole cells. The method of encapsulating whole cells in alginate beads is a cheap and simple method that enables the use of mild process conditions. Calcium alginate is one of the most commonly used carriers for the immobilization of whole cells. This method of encapsulation has several advantages, good biocompatibility, low cost, it is easily available and simple to prepare. In this work, recombinant Escherichia coli cells harboring HheC-ISM4 variant were immobilized in alginate beads and employed in rotating bed reactor (RBR) for the synthesis of (S)-2-(4-fluorophenyl)-3-hydroxypropanenitrile, valuable fluorinated building block for uses in fine chemicals industry. Given synthesis resulted in a high product yield and high optical purity of the product. The advantages of the RBR reactor over traditional batch reactor include enhancement of mass transfer, which often represents limitation in immobilized whole-cell synthesis, together with extremely simple recycling of the biocatalyst and its reuse in subsequent reactions. more...
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- 2023
15. MUZZA LAB (FKIT)
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Vuković Domanovac, Marija, Ašperger, Danijela, Matijašić, Gordana, Lučić Blagojević, Sanja, and Findrik Blažević, Zvjezdana
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radionice, dizajn prostora, Muzza Lab - Abstract
U periodu od 19. do 21. svibnja 2023. na Zagrebačkom Velesajmu, u Paviljonu 10A, održan je II. MUZZA TJEDAN ZNANOSTI. Sveučilište u Zagrebu Fakultet kemijskog inženjerstva i tehnologije kao parter ovog događaja se prezentirao s temom MI VIDIMO NEVIDLJIVO! Osmišljen je prostor (MUZZA LAB) i organizirano je 17 radionica koje je tijekom tri dana održavalo 80 nastavnika, suradnika i studenata FKIT-a. MUZZA LAB je prostor od 90 m2 koji je bio podijeljen u četiri zone (plava, zelena, crvena i narančasta). Na svakoj od četiri zone izmjenjivale su se interaktivne radionice koje su činile tematsku cjelinu i uklapale se u poruku "Mi vidimo nevidljivo". Radionice su se održavale u puni sat (10-19 sati) i trajale 30 min. Tijekom tri dana ukupno je održano 108 radionica u kojima je sudjelovalo preko 1200 sudionika u dobi od 5 do 12 pa i više godina. MUZZA LAB je održan u sklopu promicanja znanosti i Fakulteta kemijskog inženjerstva i tehnologije u STEM području. Temom "Mi vidimo nevidljivo" se željela poslati poruka da je sve oko nas složeno, slojevito, skriveno, ali ne i nedokučivo. Promotrimo svijet drugim „očima“ i otkrit ćemo njegovu ljepotu. more...
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- 2023
16. Optimization of the biocatalytic cascade synthesis of (R)-3-hydroxy-γ-butyrolactone by means of kinetic modelling
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Ćevid, Ivana, Milčić, Nevena, Majerić Elenkov, Maja, Findrik Blažević, Zvjezdana, and Rogošić, Marko
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biocatalysis ,cascade reaction ,HBL ,kinetic modelling - Abstract
Biocatalysis can be defined as the use of enzymes or whole cells to speed up synthesis reactions. [1] The newer concept in biocatalysis is the synthesis of valuable chemicals achieved through complex cascades with different enzymes sustainably and efficiently. In comparison with conventional chemical processes, cascade reactions offer many advantages like no need for isolation of intermediate products, mild and controlled reaction conditions, simple and economically profitable reaction system which is composed of one reactor. [2] 3-hydroxy-γ-butyrolactone (HBL) is a functional chiral building block with application in the synthesis of different polymers, solvents and pharmaceuticals. With an enzymatic approach, the synthesis of HBL can be economically viable while maintaining the high selectivity of this valuable chemical. [3] In this work, the kinetic model for the cascade reaction (Fig. 1) was developed. Model validation was done by performing the batch reactor experiments. Simulations were used to optimize reaction conditions and choose the best reactor design. References [1] Đ. Vasić-Rački, Z. Findrik, A. Vrsalović Presečki, Appl. Microbiol. Biotechnol. 91 (2011), 845–856. [2] M. Sudar, Z. F. Blažević, Enzyme Cascade Kinetic Modelling, in: S. Kara, F. Rudroff Enzyme Cascade Design and Modelling, Springer International Publishing, Cham, 2021, pp. 91–108. [3] H. Dhamankar, Y. Tarasova, C. H. Martin, K. L. J. Prather, Metab. Eng. 25 (2014) 72-81. more...
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- 2023
17. Slatke tajne enzima
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Milčić, Nevena, Sudar, Martina, Findrik Blažević, Zvjezdana, Bošnjak, Ivana, Çakar, Mehmet Mervan, Leaković, Emerik, Mateša, Lorena, Skendrović, Dino, Bolješić, Leo, Kos, Krešimir, and Petrić, Ivana
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enzimi, primjena enzima, razgradnja šećera - Abstract
Na ovoj radionici upoznat ćemo sudionike s dva enzima koji su vrlo zastupljeni u prehrambenoj industriji, a funkcija im je razgradnja kompleksnih šećera na one jednostavnije. U prvoj aktivnosti će se koristiti enzim laktaza za razgradnju laktoze u mlijeku na glukozu te galaktozu te određivati sadržaj nastale glukoze. U drugoj aktivnosti će se pratiti bistrenje voćnog soka te promjene na kriškama voća koje nastaju uslijed djelovanja enzima pektinaze. more...
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- 2023
18. Biocatalytic synthesis of enantiopure fluorinated building blocks: discovering the bottlenecks by enzyme reaction engineering approach
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Milčić, Nevena, Sudar, Martina, Dokli, Irena, Majerić Elenkov, Maja, Findrik Blažević, Zvjezdana, and Rogošić, Marko
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Fluorinated building blocks ,Halohydrin dehalogenase ,Enzyme kinetics ,Mathematical modelling ,Reaction engineering ,Reaction bottlenecks - Abstract
Optically pure fluorinated organic azides represent synthetically valuable building blocks in a range of industrial applications. Since the direct fluorination of molecules is challenging from both economic and environmental perspective, the development of novel methods for modifying existing fluorinated synthons is highly desirable. In this work, enantioselective azidolysis of fluorinated aromatic epoxides catalyzed by halohydrin dehalogenase (HHDH) was explored. A series of 11 fluorinated epoxides were evaluated as substrates from the viewpoint of hydrolytic stability and enzyme kinetics. Synthesis of enantiopure (R)-2-azido-1-[4-(trifluoromethyl)phenyl]ethanol with HheC-W249P variant was selected for detailed kinetic investigation. Reaction bottlenecks were identified. Epoxide hydrolysis, enzyme inhibitions and operational stability decay were found to undesirably affect the reaction outcome. Understanding the kinetic limitations and applying model-based process simulations enabled the selection of reactor type and initial conditions favoring biotransformation. By selecting a repetitive batch reactor set-up, the reaction yield of 95% could be obtained, together with the increase in the reaction selectivity of 100% compared to the batch reactor. This poster was funded by Croatian Science Foundation (IP-2018-01-4493). more...
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- 2023
19. Enzimska sinteza kiralnih građevnih blokova upotrebom halogenhidrin-dehalogenaza
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Mehić, Emina, Hok, Lucija, Findrik Blažević, Zvjezdana, Vianello, Robert, Majerić Elenkov, Maja, Majerić Elenkov, Maja, Vianello, Robert, and Kirin, Srećko I
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kiralni građevni blokovi ,biokataliza ,epoksidi - Abstract
Halogenhidrin-dehalogenaze (HHDH) su enzimi koji imaju sposobnost uvođenja novih funkcionalnih skupina u molekulu, kataliziranjem reakcije otvaranja epoksida s različitim nukleofilima. Aktivno mjesto enzima može primiti više različitih neprirodnih nukleofila poput azida, cijanida i nitrita, zbog čega su ovi enzimi privukli pažnju sintetske organske kemije. Njihova enantioselektivnost ovisna je o strukturi supstrata, te često nije dovoljno visoka za sintetsku primjenu. Enzim HheB2 iz grupe B u prethodnim istraživanjima testiran je na relativno malom broju supstrata gdje je pokazao nisku enantioselektivnost, te je dugi niz godina ostao zanemaren u biokatalitičkim istraživanjima. U okviru ovog rada provedeno je opsežno ispitivanje dva divlja tipa HHDH iz skupine B (HheB iz Croynebacterium sp. soj N-1074 i HheB2 iz Mycobacterium sp. soj GP1) na nizu strukturno različitih supstrata i nukleofila. Otkriveno je nekoliko visoko enantioselektivnih reakcija (E > 200), gdje je HheB pokazao bolju enantioselektivnost i aktivnost prema većem broju supstrata u usporedbi s HheB2. Ova dva enzima dijele visoku homologiju sekvence i razlikuju se u samo četiri aminokiseline (F36I, T120A, C124Y i H125Q). Ispitivanjem mutanata HheB2 s modificiranim pojedinačnim aminokiselinama utvrđene su aminokiseline ključne za veću enantioselektivnost HheB u odnosu na HheB2. Kombinacijom niza računalnih pristupa interpretirani su dobiveni eksperimentalni rezultati te je pokazano da su kinetički i termodinamički parametri reakcija ključni za uspješnu enantioselektivnost enzimske reakcije. Nadalje, otkriveno je da većina testiranih nukleofila uzrokuje inhibiciju enzima pri višim koncentracijama. Koristeći enzime HheB i HheB2 uspješno je provedena sinteza nekoliko β-supstituiranih alkohola i oksazolidinona s kvaternim stereocentrom u iskorištenju 37-44% i enantiomerne čistoće 94-99 % ee. more...
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- 2023
20. Potencijal biotehnologije kao klimatski neutralne tehnologije
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Findrik Blažević, Zvjezdana, Gazivoda Kraljević, Tatjana, and Hranjec, Marijana
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biotehnologija ,enzimi - Abstract
Biotehnologija je vrlo stara tehnologija. Čovječanstvo ju koristi značajno dulje od razdoblja kada ju je počelo razumijevati. Od 19. stoljeća kada je otkriveno da se fermentacija odvija zbog enzima koji se nalaze u stanicama mikroorganizama, pa sve do danas, napravljeni su značajni iskoraci u razumijevanju procesa i svega što se događa u mikroorganizmima, ali i u razvoju naprednih tehnologija i tehnikama koje su omogućile dobivanje biokatalizatora s unaprjeđenim svojstvima i u velikim količinama1. Priroda je od davnina uspješna u razvoju procesa na visokoj skali kojima rješava određene izazove u prirodi i kao takva nam može služiti kao nepresušan izvor inspiracije. Kao primjer možemo navesti fotosintezu kojom priroda neutralizira CO2 stvarajući kisik. Ipak, aktivnost u tehnosferi je prevelika, pa količina otpada i stakleničkih plinova koje čovječanstvo generira rastu. Stoga priroda treba našu pomoć kroz razvoj tehnologija s minimalnim negativnim učinkom na okoliš. Osim što se danas radi na razvoju enzima za razgradnju plastike, gdje je zabilježen značajan napredak, mnogo je i svijetlih primjera zamjene tradicionalnih kemijskih procesa biokatalitičkim procesima u industriji, a i novih procesa koji uključuju sintezu korisnih kemikalija poput lijekova (slika 1), polimera i biogoriva. Ovi su procesi bolji za okoliš, ekonomski su isplativi i troše manje energije jer se odvijaju pri blagim reakcijskim uvjetima. Strategija biogospodarstva EU2 jasno definira smjerove razvoja biotehnologije, a jedan od njih uključuje osnaživanje i uvećanje procesa u bio- sektoru. Također, prema Planu strateških inovacija i istraživanja3 smatra se da bioindustrija treba raditi na inovativnim i obećavajućim tehnologijama i dizati ju na industrijsku razinu. Među tehnologijama se navode i industrijska biotehnologija i biokataliza. Slika 1 Shematski prikaz biokatalize. 1 Editorial, Nature Catalysis 3 (2020) 179-180 2 European Commission, Directorate-General for Research and Innovation, A sustainable bioeconomy for Europe: strengthening the connection between economy, society and the environment: updated bioeconomy strategy, Publications Office, 2018, https://data.europa.eu/doi/10.2777/792130 3 European Commission, Directorate-General for Research and Innovation, Strategic Research and Innovation Agenda (SRIA) of the European Open Science Cloud (EOSC), Publications Office of the European Union, 2022, https://data.europa.eu/doi/10.2777/935288 more...
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- 2023
21. Halohydrin dehalogenase-catalysed synthesis of enantiopure fluorinated building blocks: bottlenecks found and explained by applying a reaction engineering approach
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Milčić, Nevena, primary, Sudar, Martina, additional, Dokli, Irena, additional, Majerić Elenkov, Maja, additional, and Findrik Blažević, Zvjezdana, additional
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- 2023
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22. Continuous enzymatic carboligation of benzaldehyde and acetaldehyde in an enzyme ultrafiltration membrane reactor and laminar flow microreactors
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Valinger, Davor, Vrsalović Presečki, Ana, Kurtanjek, Želimir, Pohl, Martina, Findrik Blažević, Zvjezdana, and Vasić-Rački, Đurđa
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- 2014
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23. Front Cover: Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co‐solvent on the Structural and Catalytic Properties of a Biocatalyst (Chem. Eur. J. 56/2022)
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Milčić, Nevena, primary, Stepanić, Višnja, additional, Crnolatac, Ivo, additional, Findrik Blažević, Zvjezdana, additional, Brkljača, Zlatko, additional, and Majerić Elenkov, Maja, additional
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- 2022
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24. Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co‐solvent on the Structural and Catalytic Properties of a Biocatalyst
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Milčić, Nevena, primary, Stepanić, Višnja, additional, Crnolatac, Ivo, additional, Findrik Blažević, Zvjezdana, additional, Brkljača, Zlatko, additional, and Majerić Elenkov, Maja, additional
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- 2022
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25. Imobilizacija halogenhidrin-dehalogenaze metodom kapsuliranja u metalo-organske mreže
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Milčić, Nevena, Švaco, Petra, Kovačević, Mirela, Kolić, Magdalena, Kos, Krešimir, Sudar, Martina, Vasić, Katja, Hojnik Podrepšek, Gordana, Primožič, Mateja, Leitgeb, Maja, Majerić Elenkov, Maja, Findrik Blažević, Zvjezdana, Žižek, Krunoslav, Katančić, Zvonimir, and Kovačić, Marin more...
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Halogenhidrin-dehalogenaza ,metalo organske mreže ,imobilizacija enzima - Abstract
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“. more...
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- 2022
26. Biocatalyst improvement of halohydrin dehalogenase
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Vasić, Katja, Milčić, Nevena, Findrik Blažević, Zvjezdana, Majerić Elenkov, Maja, Knez, Željko, and Leitgeb, Maja
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halohydrin dehalogenase ,magnetic nanoparticles ,functionalization ,immobilization ,cross-linking - Abstract
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. more...
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- 2022
27. Benefits of reaction engineering in biocatalysis
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Findrik Blažević, Zvjezdana, Bommarius, Andy, Mitchell, Vesna, and Fuerst, Doug
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kinetic modeling, reaction optimization, biocatalysis - Abstract
Due to its numerous benefits, biocatalysis is considered as a transformational technology for chemical production1. This does not surprise considering the mild process conditions and high catalytic power of enzymes. Still, when it comes to its application in industry, not all goes always as well as planned. Industrial biotransformations need to be set up in a way to produce significant amount of product2 with high product yield, excellent enantioselectivity, volume productivity, substrate conversion etc. Thus, a careful choice of reactor set-up, facilitated by a detailed kinetic analysis, is important.3 Nowadays, multi-step enzyme catalyzed reactions are of great importance as they facilitate a transformation with lower downstream processing expenses, equilibrium shifting as well as other economic benefits related to the usage of equipment. For an efficient enzymatic process in general, it is important to combine the knowledge of enzyme kinetics, enzyme operational stability, as well as reaction equilibrium. This reveals the dependence of crucial process variables, and enables deep analysis by using the mathematical model and a software (Fig. 1). In this work, the methodology of enzyme reaction engineering applied in our group will be presented. A focus will be put on the problems we found crucial to be investigated in complex enzyme-catalyzed reactions, such as the presence of side-reactions, poor equilibrium position and the enzyme operational stability. Positive outcomes of the application of modeling to overcome these obstacles will be presented. more...
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- 2022
28. Halohydrin dehalogenase-catalysed synthesis of (R)- epichlorohydrin
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Ćevid, Ivana, Milčić, Nevena, Majerić Elenkov, Maja, and Findrik Blažević, Zvjezdana
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Halohydrin dehalogenase ,(R)-epichlorohydrin ,kinetic characterisation ,incubation ,deactivation rate - Abstract
Enantiopure epichlorohydrin (ECH) is an important building block for the production of chiral pharmaceuticals and agrochemicals such as L- carnitine, β-blockers and pheromones. ECH is also used as an intermediate in the preparation of paper chemicals, synthetic rubbers, epoxy resins, as a starting material in the production of adhesives, surfactants, insecticides, coatings, plasticizers and solvents. Conventional ways of producing ECH have disadvantages such as relatively low reaction yields (around 50 %), high energy consumption and use of a metal catalyst (cobalt-salen complexes) that lead to environmental pollution. Because all mentioned, biocatalytic methods for the preparation of chiral ECH are getting much attention. The advantages of using biocatalytic methods are high enantioselectivity, low production cost, extensive enzyme sources and environmentally friendly conditions. Halohydrin dehalogenases (HHDHs) can be used as biocatalysts for the ring-closure of a haloalcohol to an epoxide (Figure 1). HHDHs belong to the superfamily of the short-chain dehydrogenase/reductase proteins and structurally they are homotetramers. HHDHs are divided into subtypes from letter A to G. These enzymes are easily obtained from different bacterial hosts and can be expressed in recombinant form in Escherichia coli. In this study, enzymes from Corynebacterium sp. N- 1074 (HheB) and modified HHDH from Mycobacterium sp. GP1 (HheB2-T120A), belonging to group B, were utilised for (R)-ECH synthesis (Figure 1). The kinetic characterisation was done with both enzymes and kinetic models for enzymatic synthesis of (R)-ECH from 1, 3-dichloro- 2-propanol (DCP) were developed. The kinetic characterisation is a crucial step in the development of a mathematical model as it offers a better understanding of reaction which leads to the optimization of the reaction conditions. Except kinetic characterization, the stability of enzymes during incubation with different concentrations of (R)- ECH and DCP was assessed. The experiments were done by incubating the enzymes with a different substrate (DCP) or product ((R)-ECH) concentration over 24 hours period. Enzyme activity assay was performed spectrophotometrically through absorption differences between para-nitro-2-bromo- 1- phenylethanol and para-nitrostyrene oxide. It was found that (R)-ECH has no considerable effect on enzyme stability, while DCP decreased activity of both enzymes in concentration-dependent manner. Subsequently, validation of models was performed in batch reactor experiments. As implied by monitoring enzyme stability during incubation, it was confirmed within validation experiments that substrate deactivates the enzyme. The deactivation rate constants were obtained through batch reactor experiments and it was concluded that the deactivation rate is dependent upon the concentration of the substrate. Mathematical modelling is an essential step in the optimization of a process. The model can be used to predict reaction profiles in various reactor types and under different conditions. That information can be used for selection of the appropriate reactor type and process conditions. With this approach, mathematical modelling can also be used for the scale-up of the process. Acknowledgements: Work was supported by CAT PHARMA (KK.01.1.1.04.0013), a project co-financed by the Croatian Government and the European Union through the European Regional Development Fund - the Competitiveness and Cohesion Operational Programme. more...
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- 2022
29. Green synthesis of fluorinated optically pure compound
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Marić, Ana-Katarina, Milčić, Nevena, and Findrik Blažević, Zvjezdana
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biocatalysis ,optically pure compound ,enzyme kinetics ,mathematical modelling - Abstract
Optically pure compounds are of great significance in the pharmaceutical industry, with the increasing interest in fluorinated derivatives. One important enzyme group employed in synthesizing various chiral building blocks are halohydrin dehalogenases (HHDHs). HHDHs 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. [1] The foundation of this work lies in a biocatalytic reaction shown in Fig. 1. Mathematical model for the synthesis of (S)-2-(4-fluorophenyl)-3-hydroxypropanenitrile was earlier developed by coupling kinetic equations with mass balances. 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 initial epoxide concentration was observed within incubation experiments and operational stability decay was mathematically described and included in the model. 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. Mathematical modelling has shown that synthesis in batch/repetitive batch reactor is the optimal pathway for production of this compound. more...
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- 2022
30. Assessment of C-type halohydrin dehalogenase stability
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Milčić, Nevena, Švaco, Petra, Sudar, Martina, Majerić Elenkov, Maja, and Findrik Blažević, Zvjezdana
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halohydrin dehalogenases ,operational stability ,kinetic modelling - Abstract
Halohydrin dehalogenases (HHDHs) are important enzymes that display excellent selectivity and activity in reversible dehalogenation of vicinal halohydrins. Furthermore, HHDHs may employ wide range of unnatural nucleophiles in ring-opening reactions, extending the possibilities for the synthesis of enantiopure β-alcohols1. On account of these desirable properties, the variant of the HheC enzyme is employed in the synthesis of Lipitor, one of the best-selling pharmaceuticals in the world2. However, insufficient is known about the enzyme kinetic stability that is a prerequisite for further industrial implementations2, 3. In this work, we assessed the stability wild-type HheC and two mutants with enhanced kinetic properties. First, freeze-thaw cycle testing was performed via activity measurements, whereby HheC proved to be very stable, facilitating protein handling procedures. The kinetic stability of different biocatalyst forms (whole cells, cell-free extract, and purified enzyme) in buffer was determined through activity tests, which led us to the conclusion that the cell wall and other proteins in the extract protect the enzyme and keep it active for longer period. Size distribution of purified protein was also investigated by dynamic light scattering methods to establish existence of larger protein particles formed due structural changes and aggregation. The stability of mutants during incubation with substrates and unnatural nucleophiles (azides, cyanides) was monitored to determine whether the components of the reaction mixture have an effect on enzyme activity. It was found that at higher concentrations of fluorinated styrene oxide derivatives, the enzyme loses activity faster, which was later reflected in the results of enzyme operational stability decay in the batch reactor. The co-dependency of operational stability decay rate constant on substrate initial concentration (Figure 1) was mathematically described by Eq 1. Understanding and quantifying the influence of compounds concentrations on enzyme behavior is of a great importance for larger scale processes3. Presented results give a better insight into the limitations of HheC application and help with biocatalysts handling for prolonging its longevity in reactors. more...
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- 2022
31. Evaluacija nitrilaza kao biokatalizatora za sintezu (R)-4-kloro-3-hidroksibutanske kiseline
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Ćevid, Ivana, Antolović, Ana, Milčić, Nevena, Majerić Elenkov, Maja, Findrik Blažević, Zvjezdana, Žižek, Krunoslav, Katančić, Zvonimir, and Kovačić, Marin
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Biokatalizator ,nitrilaza ,evaluacija ,(R)-4-kloro-3-hidroksibutanska kiselina - Abstract
Enzimi nitrilaze, bez stvaranja slobodnih amidnih međuprodukata, kataliziraju reakcije hidrolize nitrila u karboksilne kiseline i amonijak. [1] Stoga se nitrilaza može koristiti kao biokatalizator u reakcijama pretvorbe (R)-4-kloro- 3-hidroksibutironitrila (KHB) u (R)-4-kloro-3- hidroksibutansku kiselinu, pri čemu dolazi do spontane ciklizacije produkta u (R)-3-hidroksi-γ- butirolakton (HBL) (slika 1). HBL je koristan kiralni građevni blok s primjenom u sintezi različitih polimera, otapala i farmaceutika. Postoje brojni kemijski putevi sinteze HBL-a, no većina ima nedostatke poput skupih ili opasnih reaktanata, intenzivnih procesnih uvjeta, te niskih iskorištenja i selektivnosti, što uzrokuje poskupljenje procesa izdvajanja produkta. Nasuprot tome, enzimski katalizirani procesi koriste blage reakcijske uvjete, postižu visoku selektivnost te mogu pružiti ekonomski isplativ postupak sinteze ove vrijedne kemikalije. [2, 3] U ovom radu provedena je evaluacija 24 nitrilaze. Tijekom ispitivanja korišteni su isti početni uvjeti, koncentracija supstrata (KHB) i masena koncentracija nitrilaza, dok se reakcija pratila 6 sati. Enzim s najvećim iskorištenjem na produktu izabran je za sljedeći korak, kinetičko istraživanje. Određeni su kinetički parametri nitrilazom katalizirane sinteze (R)-4-kloro-3- hidroksibutanske kiseline iz (R)-4-kloro-3- hidroksibutironitrila. more...
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- 2022
32. APPLICATION OF REACTION ENGINEERING IN BIOCATALYSIS
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Findrik Blažević, Zvjezdana and Sauer, Michael
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kinetic modelling, reaction optimization, biocatalysis - Abstract
Due to its numerous benefits, biocatalysis is considered as a transformational technology for chemical production1. This does not surprise considering the mild process conditions and high catalytic power of enzymes. Still, when it comes to its application in industry, not all goes always as well as planned. Industrial biotransformations need to be set up in a way to produce significant amount of product2 with high product yield, excellent enantioselectivity, volume productivity, substrate conversion etc. Thus, a careful choice of reactor set-up, facilitated by a detailed kinetic analysis, is important.3 Nowadays, multi-step enzyme catalyzed reactions are of great importance as they facilitate a transformation with lower downstream processing expenses, equilibrium shifting as well as other economic benefits related to the usage of equipment. For an efficient enzymatic process in general, it is important to combine the knowledge of enzyme kinetics, enzyme operational stability, as well as reaction equilibrium. This reveals the dependence of crucial process variables, and enables deep analysis by using the mathematical model and a software (Fig. 1). In this work, the methodology of enzyme reaction engineering applied in our group will be presented. A focus will be put on the problems we found crucial to be investigated in complex enzyme-catalyzed reactions, such as the presence of side-reactions, poor equilibrium position and the enzyme operational stability. Positive outcomes of the application of modeling to overcome these obstacles will be presented. more...
- Published
- 2022
33. Fakultet kemijskog inženjerstva i tehnologije, Samoanaliza 2022
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Jukić, Ante, Bolf, Nenad, Findrik Blažević, Zvjezdana, Lučić Blagojević, Sanja, Bolanča, Tomislav, Gazivoda Kraljević, Tatjana, Kušić, Hrvoje, Lončarić Božić, Ana, Rogošić, Marko, Kurajica, Stanislav, Matijašić, Gordana, Otmačić Ćurković, Helena, Kassal, Petar, Kučić Grgić, Dajana, Faraguna, Fabio, Ukić, Šime, Katančić, Zvonimir, and Sejdić, Marko more...
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Fakultet kemijskog inženjerstva i tehnologije, Sveučilište u Zagrebu, samoanaliza - Abstract
Ovo je službeni dokument, samoanaliza Fakulteta kemijskog inženjerstva i tehnologije Sveučilišta u Zagrebu za reakreditaciju u 2022.
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- 2022
34. Radionice 2
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Vuković Domanovac, Marija, Findrik Blažević, Zvjezdana, Stojmilović, Ivana, Trtinjak, Martina, Ašperger, Danijela, Babić, Bruna, Mužina, Katarina, and Brleković, Filip
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mikroorganizmi, čokolada, nano materijali - Abstract
U sklopu promicanja znanosti i Fakulteta kemijskog inženjerstva i tehnologije Sveučilišta u Zagrebu u STEM području u OŠ Pavleka Miškine Zagreb u Zagrebu održane su tri radionice: 1. ŽIVOT U KAPLJICI VODE, voditeljice: Marija Vuković Domanovac, Zvjezdana Findrik Blažević, Ivana Stojmilović i Martina Trtinjak ; 2. ČOKOLADA=VALOVI UŽITKA, voditeljice: Danijela Ašperger i Bruna Babić ; 3. SILIKATI, OKSIDI ILI DRAGULJI MALI, SVE SU TO (NANO)MATERIJALI, voditelji: Katarina Mužina i Filip Brleković. more...
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- 2022
35. Biocatalytic properties of group B halohydrin dehalogenases in nucleophilic ring-opening reactions of epoxides
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Mehić, Emina, Findrik Blažević, Zvjezdana, and Majerić Elenkov, Maja
- Subjects
Biocatalyst, HHDH, enantioselectivity - Abstract
Halohydrin dehalogenases (HHDHs) belong to a distinct group of enzymes catalyzing the removal of a halide ion and a proton from a vicinal halohydrin with the formation of an epoxide. Moreover, these enzymes can catalyze the epoxide ring-opening reactions with a range of anionic nucleophiles affording β-substituted alcohols as products. Based on sequence similarities, they are divided into 7 phylogenetic groups: A, B, C, D, E, F and G. Activity, enantioselectivity and enantiopreference are dependent on the type of enzyme and the substrate structure. HheC from Agrobacterium radiobacter is the most studied because of its high enantioselectivity. In order to expand the catalytic relevance of HHDHs we focus our attention to an enzyme from B group on which a small amount of research has been done. There are two similar enzymes in group B, one found in Corynebacterium sp. N1074 (HheB) and the other in Mycobacterium sp. GP1 (HheB2). They share high sequence identity (95%) with only 4 amino acid substitutions HheB/HheB2: F36/I36, T120/A120, C124/Y124, H125/Q125. HheB exhibits higher enantioselectivity than does HheB2. HheB2 has been previously characterized as a non-enantioselective enzyme, and neglected as a biocatalyst. In this work, HheB2 and HheB were investigated in the ring-opening reaction on a set of 21 structurally different aliphatic and aromatic epoxides using sodium azide as a nucleophile. To gain more insight into the difference of their enantioselectivity, mutants HheB2-F36I, HheB2- T120A, HheB2-C124Y, HheB2-H125Q were constructed and also tested. We examined nucleophiles that are accepted in the active site of B-type and used kinetics measurements to determine enzyme affinity and inhibition concentration for all accepted nucleophiles. The screening confirmed low to moderate enantioselectivity of HheB2 towards monosubstituted epoxides, however high activity and enantioselectivity in conversion of 2, 2- disubstituted (E-values up to >200). Enantioselectivity of HheB was slightly higher than HheB2 in the majority of cases. T120A mutation has been shown to be a key for increasing enantioselectivity in aliphatic epoxides. Among all tested substrates, disubstituted benzyl epoxides showed great potential for producing enantiomerically pure β-substituted alcohols. We showed that bicyclic structures as well, can be accepted into the active site of B-type with high E values (>200). Screening of nucleophiles confirmed that azide, cyanide, nitrite, cyanate and thiocyanate are accepted into the active site of the enzyme. A kinetics measurement showed that reaction rates are highest with azide ion and it is the only nucleophile that does not cause enzyme inhibition even at concentrations of 100 mM. Four other nucleophiles (CN-, NO2-, OCN- and SCN-) cause inhibition at concentrations between 5 and 25 mM. more...
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- 2022
36. Elucidation of DMSO effects on catalytic activity of halohydrin dehalogenase HheC by molecular dynamics
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Stepanić, Višnja, Brkljača, Zlatko, Milčić, Nevena, Crnolatac, Ivo, Findrik Blažević, Zvjezdana, Majerić Elenkov, Maja, and Hungarian Biophysical Society
- Subjects
halohydrin dehalogenase, biocatalysis, dimethyl sulphoxide, molecular dynamics, DLS, DSC - Abstract
Homotetrameric halohydrin dehalogenase from Agrobacterium radiobacter AD1, HheC is extensively used for industrial green synthesis of enantiopure building blocks. It naturally catalyses reversible dehalogenation of vicinal haloalcohols, but it is utilized with a whole range of unnatural nucleophiles in epoxide ring-opening reactions. In order to increase solubility of lipophilic epoxides and conversion efficiency, addition of various solvent is explored. The results of our study of effects of widely explored solvent DMSO (dimethyl sulfoxide) on catalytic activity of HheC will be presented. Besides determination of kinetic parameters, differential scanning calorimetry (DSC) and dynamic light scattering (DLS), molecular dynamics (MD) is used to elucidate mechanisms of DMSO action on HheC. We carried out MD simulations (GROMACS) on natural tetrameric and hypothetical monomeric HheC in water as well as in 20% and 50% (v/v) DMSO/aqueous environment. The tetramer HheC exhibits remarkable conformational tolerance towards DMSO up to 30% and it instantly aggregates at 50% DMSO, but its catalytic activity exponentially decreases with DMSO addition. 5% DMSO inhibits the HheC activity by half. The MD demonstrates that while subunit conformations slightly changes with DMSO addition, distinct sheering of the main structural motifs between subunits occurs, with changes proceeding from more localized (20%) to more extended and collective (50%). However, no dissociation (up to 300 ns) was observed in accordance with DSC and DLS results, but buried surface area increases and the catalytic site becomes more constrained. DMSO is found to replace H2O molecules in the catalytic site forming alternately H-bonds with the catalytic amino acid residues S132 and Y145, and to form small clusters around the protein (Fig. 1). more...
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- 2022
37. Biocatalytic synthesis of enantioenriched product – reaction engineering approach
- Author
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Milčić, Nevena, Marić, Ana-Katarina, Sudar, Martina, Majerić Elenkov, Maja, and Findrik Blažević, Zvjezdana
- Subjects
fluorinated building blocks ,halohydrin dehalogenases ,enantioselectivity ,enzyme kinetics ,mathematical modelling ,process simulations - Abstract
Optically pure fluorinated organic nitriles are synthetically valuable building blocks in the pharmaceutical and fine chemical industries. Since direct fluorination is challenging from both economic and environmental perspectives, the development of methods to modify existing fluorinated synthons is a prized alternative. Halohydrin dehalogenases (HHDHs) are synthetically valuable enzymes because they can accept numerous anionic nucleophiles such as cyanide, cyanate, azide and others in epoxide ring-opening reactions. HHDHs exhibit enantioselectivity or enantiopreference in a wide range of reactions and provide the ability to perform kinetic resolution of fluorinated building blocks, resulting in higher value products, i.e., optically pure β-substituted alcohols and unreacted enantiomers of substrates. However, if the enzyme is not completely enantioselective, the optical purity of the final products depends largely on the reaction conditions and reactor design. In this work, the HHDH-catalysed synthesis of (S)-3-(4-fluorophenyl)-3-hydroxypropanenitrile was investigated from a reaction engineering point of view. Since both enantiomers of 2-(4-fluorophenyl)oxirane are accepted in the biocatalytic reaction with cyanide ions, the reaction was kinetically studied in detail starting from the individual enantiomers of the substrate. In addition, the inevitable hydrolysis of the substrate was kinetically characterised. The effect of all concentrations of the reaction components on the enzyme activity was investigated. Differences were found between the enzyme affinities toward (R)- and (S)-2-(4-fluorophenyl)oxirane, as well as the presence of different inhibitions in both biocatalytic reactions. These effects are expressed numerically by Vm, Km and Ki values. In addition to activity, the stability of the enzyme in the presence of compounds from the reaction mixture was also evaluated, and as a result, a co-dependence of the operational stability decay on the initial substrate concentration was found. The mathematical model for the synthesis of (S)-3-(4-fluorophenyl)-3-hydroxypropanenitrile was developed and used as a guide for the detection and minimization of reaction bottlenecks. Through mathematical modelling, it is possible to manipulate the outcome of the reaction in the desired direction within the limitations of the enzyme. Process simulations were used to select suitable conditions for the synthesis of a product with high optical purity and yield. more...
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- 2022
38. Current and future mobility and networking opportunities in Croatia for students and ECI researchers
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Panžić, Ivana, Mandić, Vilko, and Findrik-Blažević, Zvjezdana
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mobility ,students ,ECI - Abstract
The current state of mobility in Croatia, in both the outgoing and incoming student mobility numbers is at a lower level than other EU countries. The numbers can and must be improved in the next years. One way to tackle this problem is to increase the visibility of Croatian institutes and Universities by using more opportunities like conferences, workshops and other networking events and initiatives. University of Zagreb, including faculties like Faculty of Chemical Engineering and Technology and Faculty of Science have a lot to offer especially in the field of chemistry and material sciences in general. Despite the lacking infrastructure and smaller number of large EU projects we believe that visiting students (both masters and PhD level) and young researchers (postdocs) could benefit from a short term scientific visit to FCET, additionally leading to a two-way knowledge transfer, new collaborations and successful future project applications in the field of the Green Field EU initiative. Also, the broadening of the current collaborations would make it easier for Croatian students to find either PhD or postdoc positons in EU, after which they could return to Croatia and form their own group easier. more...
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- 2022
39. Showcase for material science R&D at FCET for increasing participation in EU funding schemes
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Mandić, Vilko, Panžić, Ivana, and Findrik-Blažević, Zvjezdana
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material science ,R&D ,EU funds ,green deal - Abstract
Faculty hosts several smaller younger research groups which already developed high national prominence. Reaching out to EU level of prominence proved difficult primarily due to lack of wider participation in the EU funding schemes. That however proved primarily to be the consequence of limited development of Croatian research infrastructure. Therefore, for a Croatian applicant getting an ERC grant is hardly possible, because the EU basically presumes that you have the necessary infrastructure, and does not really care is your origin is systematically lagging behind. During the whole ERC program, 2 ERC starting grants (from the cca 7000 ERC grants 2007–2022 in PE) were received in Croatia, to prove my point. The ideas behind the research activities of the prominent groups from FCET focus broadly between the fundamental phenomena and tasks resolving the state-of-the-art and beyond technical challenges. Again the bottlenecks are found within instrumental requirements. My idea is to present the interesting expertise from the FCET in terms of specific, and novel synthesis, deposition, characterisation and testing setups. On behalf of bilateral or even multilateral communication of should be possible to elucidate overlap of interest and identify opportunities to resolve issues. Provided the meeting raises awareness on the suitable EU finding program, in conclusion it should be possible to foster increased participation in such funding by Croatian researchers as well. The things to consider for fostering greater utilisation of EU funding are broadening of infrastructure, utilisation of their access, keeping up with the maintenance, managing administration and the burden of bureaucratic necessities, etc. Particularly interesting would be to participate in specific ambitious work environment such as networking sessions to maximise the collaboration opportunities. As a consequence, excellent researchers with high driving force should have more easy time to remain in Croatia and continue building their groups without the fear of reaching the (now obvious) participation limits. more...
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- 2022
40. Cascade Synthesis of l-Homoserine Catalyzed by Lyophilized Whole Cells Containing Transaminase and Aldolase Activities: The Mathematical Modeling Approach
- Author
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Česnik Katulić, Morana, primary, Sudar, Martina, additional, Hernández, Karel, additional, Qi, Yuyin, additional, Charnock, Simon J., additional, Vasić-Rački, Đurdica, additional, Clapés, Pere, additional, and Findrik Blažević, Zvjezdana, additional more...
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- 2021
- Full Text
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41. Biocatalytic scope of halohydrin dehalogenase from Mycobacterium sp. GP1 (HheB2) and mutant HheB2- T120A
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Mehić, Emina, Findrik Blažević, Zvjezdana, Majerić Elenkov, Maja, Marković, Dean, Meštrović, Ernest, Namjesnik, Danijel, and Tomašić, Vesna
- Subjects
biocatalyst ,HHDH ,enantioselectivity - Abstract
Halohydrin dehalogenases (HHDHs) belong to a distinct group of enzymes catalyzing the removal of a halide ion and a proton from a vicinal halohydrin with formation of an epoxide. Moreover, these enzymes can catalyze the epoxide ring- opening reactions with a range of anionic nucleophiles affording β-substituted alcohols as products. Based on sequence similarities, they are divided into 7 phylogenetic groups: A, B, C, D, E, F and G. Activity, enantioselectivity and enantiopreference are dependent on the type of enzyme and the substrate structure. Among all, the enzyme HheC from Agrobacterium radiobacter (group C) is the most studied because of its high enantioselectivity and wide nucleophile scope. However, the major shortcoming of HheC is its narrow substrate tolerance, due to the relatively small active site. To expand the catalytic relevance of HHDHs we focus our attention to an enzyme from different organism. There are two similar enzymes in group B, one found in Corynebacterium sp. N1074 (HheB) and the other in Mycobacterium sp. GP1 (HheB2).They share high sequence identity (95%) with only 4 amino acid substitutions HheB/HheB2: F36/I36, T120/A120, C124/Y124, H125/Q125. HheB exhibits higher enantioselectivity than does HheB2. Because of that HheB2 has been previously characterised as non-enantioselective enzyme, and neglected as biocatalyst. In this work HheB2 was investigated in the ring-opening reaction on a set of 21 structurally different aliphatic and aromatic epoxides using sodium azide as nucleophile. In order to gain more insight into the difference in enantioselectivity of HheB2 and HheB, mutant HheB2-T120A was also investigated in the ring opening reactions. The screening confirmed low to moderate enantioselectivity of HheB2 towards monosubstituted epoxides, however high activity and enantioselectivity in conversion of 2, 2- disubstituted (E-values up to >200). Similar to HheC, the large enhancement of enantioselectivity is obtained when a second substituent (methyl or ethyl) is present at the chiral centre. T120A mutation has been shown to be a key mutation for increasing enantioselectivity in aliphatic epoxides, which is not the case for aromatic epoxides. The results further extend the repertoire of enantioselective HHDHs and their application in the kinetic resolution of epoxides. more...
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- 2021
42. Halohydrin dehalogenase-catalysed synthesis of fluorinated aromatic chiral building blocks
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Dokli, Irena, Milčić, Nevena, Marin, Petra, Sudar, Martina, Findrik Blažević, Zvjezdana, and Maja Majerić Elenkov
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biocatalysis ,halohydrin dehalogenase ,epoxide ,kinetic resolution - Abstract
Halohydrin dehalogenases (HHDHs) catalyse enantioselective formation and conversion of epoxides. They can be applied in the kinetic resolution of racemic epoxides by utilising a range of anionic nucleophiles such as azide, cyanide, cyanate or nitrite.[1, 2] The ring- opening products and remaining epoxides are versatile and important synthetic intermediates for fine chemicals. In this work HHDH was employed for the transformation of epoxides bearing fluoroaryl groups (Figure 1). Fluoroaromatic compounds are widely used as starting materials for chemical syntheses, especially pharmaceuticals and agrochemicals. Introduction of fluorine atoms into the molecule usually increases target effectiveness, biological half-life, bioabsorption etc. The growing interest in fluorinated organics makes the development of synthetic procedures leading to such compounds desirable. Kinetic resolution of a series of fluorinated styrene oxide derivatives was studied using the enzyme from Agrobacterium radiobacter AD1 (HheC) and a variant W249P.[3] A mutant HheC-W249P catalysed nucleophilic ring-opening with azide and cyanide ions with excellent enantioselectivity (Evalues up to >200) and higher activity compared to the wild- type, which gives access to various enantiopure β- substituted alcohols and epoxides. It was found that the enzyme tolerates substrates in concentrations over 50 mM. However, different side reactions were observed at elevated concentrations and with prolonged reaction time. The biocatalytic azidolysis and cyanolysis of racemic 4-CF3-styrene oxide were performed on preparative scale, affording (R)-2-azido-1-(4-trifluoromethylphenyl)- ethanol in 38% yield and 97% ee, and (S)-3- hydroxy-3-(4-trifluoromethylphenyl)-propionitrile in 30% yield and 98% ee. more...
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- 2021
43. Validation of kinetic model of halohydrin dehalogenase-catalysed synthesis of (R)- epichlorohydrin
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Ćevid, Ivana, Milčić, Nevena, Majerić Elenkov, Maja, Findrik Blažević, Zvjezdana, Marković, Dean, Meštrović, Ernest, Namjesnik, Danijel, and Tomašić, Vesna
- Subjects
validation ,halohydrin dehalogenase ,(R)-epichlorohydrin - Abstract
Epichlorohydrin (ECH) is a valuable product that is commonly used as an intermediate in the preparation of synthetic rubbers, as a starting material in the production of insecticides, pharmaceuticals, etc. [1] This prominent product can be obtained through biocatalysis. Halohydrin dehalogenases (HHDHs) are adaptable and important biocatalysts that are mostly used for the industrial synthesis of enantiopure ECH. [2] The reasons why the biocatalytic approach takes precedence over the traditional one are high enantioselectivity, low production cost, extensive enzyme sources and environmentally friendly conditions of this synthesis. [3] The operational stability of an enzyme generally relates to the retention of activity when an enzyme is in use. On an industrial level, the long-term operational stability of biocatalysts is of crucial importance for deciding whether a process can be viable as opposed to the traditional approach. [4] Operational stability is necessary to determine at which process conditions enzyme can work properly. In this work the kinetic model of HheB2-T120A- catalysed synthesis of (R)-ECH from 1, 3-dichloro- 2-propanol was developed. Model validation was performed in batch reactor experiments. It was found that a substrate (1, 3-dichloro-2-propanol) deactivates the enzyme and the rate of deactivation is dependent upon its concentration. ACKNOWLEDGEMENTS: This work was supported by CATPHARMA (KK.01.1.1.04.0013). REFERENCES [1] Y. Bespalko et al., International Journal of Chemical Kinetics 2021, 53(3), 356-368. [2] Z. Findrik Blažević et al., Advanced Synthesis and Catalysis 2021, 363(2), 388-410. [3] X. J. Zhang et al., Bioresource Technology 2018, 263, 483-490. [4] G. A. Drago, T. D. Gibson, Enzyme Stability and Stabilisation: Apllications and Case Studies, Engineering and Manufacturing for Biotechnology 2002, 4, 361-376. more...
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- 2021
44. Kinetic model of halohydrin dehalogenase-catalysed synthesis of (R)-ECH
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Ćevid, Ivana, Milčić, Nevena, Majerić Elenkov, Maja, and Findrik Blažević, Zvjezdana
- Subjects
Halohydrin dehalogenase ,(R)-epichlorohydrin - Abstract
Halohydrin dehalogenases (HHDHs) are valuable and versatile biocatalysts. They can easily be obtained in large quantities by expression in recombinant E. coli cells1. HHDH enzymes are divided into subtypes A - G and show promising properties in stereoselective biocatalysis but are still insufficiently investigated1. HHDHs are industrially applied for the synthesis of chiral epichlorohydrin (ECH). ECH is widely used as a starting material in the production of insecticides, surfactants, adhesives, pharmaceuticals, etc1. Main disadvantages of the traditional synthesis of chiral ECH is use of environmentally unfriendly, metal catalyst (cobalt-salen complexes) and relatively low reaction yield as it is limited at 50 %. Biocatalytic methods for chiral ECH preparation are getting much attention mostly because of environmentally friendly conditions, extensive enzyme sources, high enantioselectivity and low production cost2. Several studies involving the synthesis of chiral ECH by HHDH mutants have been published3-5. We have developed kinetic model of HheB2-T120A-catalysed synthesis of (R)-ECH from 1, 3-dichloro-2-propanol. Kinetic characterisation offers better understanding of reaction and is an essential step in the development of mathematical model which ultimately helps with the optimization of the reaction conditions. more...
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- 2021
45. Activity and operational stability of halohydrin dehalogenases in aqueous-organic biphasic systems
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Milčić, Nevena, Sudar, Martina, Findrik Blažević, Zvjezdana, and Majerić Elenkov, Maja
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halohydrin dehalogenase ,aqueous-organic system ,operational stability - Abstract
Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that show exceptional selectivity, activity, and versatility regarding accepting nucleophiles in epoxide ring-opening reactions.[1] HHDHs can be employed in the synthesis of important and versatile pharmaceutical building block (S)-2-(4- fluorophenyl)-3-hydroxypropanenitrile through kinetic resolution of rac-2-(4- fluorophenyl)oxirane.[1, 2] The major limitations of this reaction are poor substrate solubility and its susceptibility to hydrolytic decomposition. Introduction of organic phase in the aqueous reaction media can have a positive effect both on the solubility and hydrolytic stability of epoxide, leading to higher volume productivity. However, possible side effects of the organic phase introduction are enzyme activity and operational stability decrease, hence they need to be experimentally investigated and quantified. In this work, we determined the activity of wild-type HheC from Agrobacterium radiobacter in the presence of a wide solvent/buffer ratio range. As a result, correlation between solvent logP values and concentration of half-inactivation (C50) was observed, whereby C50 represents concentration of solvent that irreversibly reduces enzyme initial activity by half.[3]In addition to affecting the initial enzyme activity, the organic solvent may also affect the enzyme activity during continuous use. Hence, enzyme stability in selected solvents and at different ratios during time was monitored. Besides HheC, we examined the co-solvent activity and stability of thermostable variant ISM-4 obtained with iterative saturation mutagenesis, [4] as resistance to high temperatures is often accompanied with enhanced co-solvent activity and stability.[5] Finally, based on the experiments performed on a model system, we investigated the co-solvent operational stability of ISM-4 during (S)-2-(4-fluorophenyl)-3-hydroxypropanenitrile synthesis. We continually monitored the enzyme activity during reaction and estimated the values of enzyme deactivation rate constant (kd, min-1). References [1] Z. Findrik Blažević ; N. Milčić ; M. Sudar ; M. Majerić Elenkov ; Adv. Synth. Catal. 2021, 363 (2), 388-410. [2] I. Dokli ; N. Milčić ; P. Marin ; M. Svetec Miklenić ; M. Sudar ; L. Tang ; Z. Findrik Blažević ; M. Majerić Elenkov ; Catal. Commun. 2021, 152, 106285. [3] V. Stepankova ; S. Bidmanova ; T. Koudelkova ; Z. Prokop ; R. Chaloupkova ; J. Damborsky ; Adv. Synth. Catal. 2013, 3 (12), 2823-2836. [4] Z. Wu ; W. Deng ; Y. Tong ; Q. Liao ; D. Xin ; H. Yu ; J. Feng ; L. Tang ; App. Microbiol. Biotechnol. 2017, 101, 3201–3211. [5] H. Arabnejad ; M. Dal Lago ; P. A. Jekel ; R. J. Floor ; A-M. W. H. Thunnissen ; A. C. T. van Scheltinga ; H. J. Wijma ; D. B. Janssen ; Protein. Eng. Des. Sel. 2017, 30 (3), 173-187. more...
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- 2021
46. Kinetic characterization of C-type halohydrin dehalogenase in the synthesis of enantiopure fluoroaromatic compounds
- Author
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Milčić, Nevena, Sudar, Martina, Findrik Blažević, Zvjezdana, and Majerić Elenkov, Maja
- Subjects
halohydrin dehalogenase ,fluorinated chiral building blocks ,kinetic mathematical modelling - Abstract
Synthesis of fluorinated aromatic compounds is of increasing interest in the pharmaceutical and agrochemicals industry. More environmentally friendly biocatalytic modification of simpler molecules that contain a C-F bond is an attractive alternative to the often expensive and toxic metal-catalyzed fluorination of molecules, especially when optically active substances are obtained through the employment of enantioselective enzymes. Halohydrin dehalogenases (HHDHs) are industrially relevant enantioselective enzymes that catalyse the reversible conversion of vicinal halohydrins to epoxides. HHDH enzymes have been widely used in cascade reaction systems, whereby the most famous example is the joint action with nitrilase for the commercialized multi-ton-scale synthesis of ethyl (R)-4-cyano-3- hydroxybutyrate, chiral key precursor for statin side chains. In this work, reactions of fluorine- substituted aryl oxyranes with sodium azide catalyzed by C type HHDH (HheC) were studied. Obtained enantiopure fluoroaromatic vicinal azidoalcohols and epoxides are valuable chiral building blocks with a wide range of applications in the design and development of new drugs. The application of mathematical modelling approach can be of great importance since it can greatly accelerate the development of a new process where time is a key parameter. Therefore, in this work, kinetic characterization of HheC enzymes in the presence of individual epoxides was performed to find the system with the maximum potential for high yield enantioselective conversion. Based on kinetic constants, a system for further characterization and development of a complete kinetic model was selected. Due to the low solubility of epoxides and the influence of chemical hydrolysis in aqueous medium, the application of aqueous-organic systems has been investigated. Their applicability was examined by monitoring the activity of enzyme in the presence of different concentrations of selected organic solvents. more...
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- 2021
47. Solvent tolerance of halohydrin dehalogenases
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Milčić, Nevena, Sudar, Martina, Findrik Blažević, Zvjezdana, Majerić Elenkov, Maja, Marković, Dean, Meštrović, Ernest, Namjesnik, Danijel, and Tomašić, Vesna
- Subjects
halohydrin dehalogenases ,solvent engineering ,thermostable enzyme variant - Abstract
Halohydrin dehalogenases catalyze reversible conversion of epoxides to β-haloalcohols in enantioselective fashion with an extensive scope of unnatural nucleophiles.[1] Selectivity and diversity of HHDH enzymes are promising yet insufficient properties to rely solely on them for industrial application purposes. Aqueous solution as physiological medium is the most convenient concerning enzyme natural properties, but poor solubility and hydrolytic instability of hydrophobic substrates make these reactions limited and unfavourable.[2] Thus, biotransformation scale-up often requires modification of the reaction medium by introduction of organic solvents (OSs), which can minimize these problems but also decrease enzyme activity and stability. As a HHDH representative, HheC from Agrobacterium radiobacter AD1 was selected and enzyme activity was determined in the presence of a wide OSs range. Correlation between OSs properties (logP, pKa) and concentration of half-inactivation (C50) was observed. To gain insight into the enzyme behavior in an alternative medium over an extended period of time, activity during incubation with selected OSs was monitored. Besides HheC, we examined the co-solvent stability during incubation of thermostable variant ISM-4. [3] Results obtained reveal that this enzyme is a robust and powerful ally for synthesis in more harsh conditions and a promising candidate for further experimentation in the direction of industrial implementations. more...
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- 2021
48. Development of biocatalytic reactions – an engineering approach
- Author
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Findrik Blažević, Zvjezdana
- Subjects
kinetic model ,experimental design ,enzyme reactions - Abstract
Biocatalytic reactions provide great opportunities in the synthesis of novel compounds and greener ways to produce the already existing ones [1, 2], which is important nowadays when we want to be less dependent upon non-renewable resources. Still, to broaden the industrial application of biochemical reactions, there are many hurdles along the way. These require resolving by an integral and interdisciplinary approach including organic chemistry, enzyme, medium, reaction and process engineering etc. [3]. The latter two approaches enable choosing the right reactor mode, as well as optimization of the reaction conditions that facilitate obtaining the process metrics goals such as product concentration and yield, volume productivity and biocatalyst yield. Considering that these process values are of crucial importance for evaluation of process viability, they represent an important milestone in any process development, on lab scale and beyond [4]. In this lecture, a special focus is placed on process/reaction engineering application in complex biocatalytic processes [5]. This area of research, even though growing in the last decade, is still not applied enough in the development of biocatalytic processes. Advantages of their applications will be presented based on experiences in our research group. [1] S. Wu, R. Snajdrova, J.C. Moore, K. Baldenius, U.T. Bornscheuer, Biocatalysis: Enzymatic Synthesis for Industrial Applications, Angew. Chem. Int. Ed. 2021, 60, 88 –119. [2] E.L. Bell, W. Finnigan, S.P. France, A.P. Green, M.A. Hayes, L.J. Hepworth, S.L. Lovelock, H. Niikura, S. Osuna, E. Romero, K.S. Ryan, N.J. Turner, S.L. Flitsch, Biocatalysis, Nature reviews, Methods Primers (2021) 1:46. [3] R.A. Sheldon, P.C. Pereira, Biocatalysis engineering: the big picture, Angew. Chem. Int. Ed. 2021, 60, 88–119. [4] P. Tufvesson, J. Lima-Ramos, M. Nordblad, J.M. Woodley, Guidelines and Cost Analysis for Catalyst Production in Biocatalytic Processes, Organic Process Research & Development 2011, 15, 266–274. [5] M. Sudar, Z. Findrik Blažević, Enzyme Cascade Kinetic Modelling, Enzyme Cascade Design and Modelling, S. Kara, F. Rudroff (Eds.), Switzerland: Springer International Publishing, 2021. p 91-108. more...
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- 2021
49. Application of a mathematical model for the optimization of a multi-enzymatic reaction; The case study of L-homoserine synthesis
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Česnik, Morana, Sudar, Martina, Hernandez, Karel, Clapes, Pere, Charnock, Simon, Vasić-Rački, Đurđa, Findrik Blažević, Zvjezdana, Rother, Dörte, and Sehl, Torsten
- Subjects
cascade reaction ,L-homoserine ,mathematical model ,optimization - Abstract
Cascade multi-step reactions have gained a lot of attention in the last decade due to their numerous advantages against traditional organic synthesis methods. However, the number of dependencies between different variables increases with the number of enzymes and makes cascades highly complex to investigate, optimize and carry out. Consequently, for industrial application, it is important to understand their complexity from the viewpoint of enzyme reaction engineering. In this work the modelling methodology was applied on the synthesis of L-homoserine, which is an important intermediate and a building block in chemical and pharmaceutical industry. Its cascade synthesis catalyzed by an aldolase and transaminase in one pot starting from pyruvate, formaldehyde and L- alanine, was investigated in detail (Scheme 1), with enzymes, as well as whole lyophilized E. coli cells as catalysts. Scheme 1 The reaction scheme of the synthesis of L-homoserine. Reactions catalyzed by enzymes as cell free extracts (CFE) and E. coli cells containing the same co-expressed enzymes were optimized based on the developed mathematical model. The model was also used for the selection of the proper reactor set-up. Optimized reaction was carried out in the fed- batch reactor with continuous formaldehyde feed and two additions of L-alanine and pyruvate. The results were not only reproducible in our lab, but also in the lab of a project partner, and up- scaled experiments confirmed the applicability and potential of developed mathematical model for L- homoserine synthesis. The modeling approach enabled obtaining 672 mM of L-homoserine and volume productivity of 76.23 g L–1 d–1 after 25 hours in the experiment when CFE-enzymes were used as biocatalysts. Similar results were obtained in the experiment with lyophilized whole cells ; i.e. 640.74 mM of L- homoserine and volume productivity of 62 g L–1 d– 1. Obtained process metrics present an excellent result with a potential for an industrial use. Project CARBAZYMES-This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 635595. more...
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- 2021
50. Simulation of a two-step cascade reaction to yield (R)-4-chloro-3-hydroxybutyronitrile
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Ćevid, Ivana, Milčić, Nevena, Majerić Elenkov, Maja, and Findrik Blažević, Zvjezdana
- Subjects
Cascade reaction ,(R)-4-chloro-3-hydroxybutyronitrile ,simulation - Abstract
In comparison with traditional organic synthesis methods, cascade multi-step biocatalytic reactions have many advantages. A few of them are sustainability, simple and cheaper reactor set- up, mild reaction conditions and lower environmental impact. [1, 2] Advancements in biocatalytic processes are achieved through reaction engineering. Combined kinetic and reactor modelling can significantly contribute to the choice of reactor mode, design and optimal operating conditions. [3, 4] Using modelling and simulations helps us study the effects of different variables on the process, which increase our knowledge and understanding of the system. [5] In this work, a two-step cascade reaction was studied (Figure 1). Both steps are catalysed by halohydrin dehalogenase (HheB2), mutant T120A. The first reaction was a ring closure of a substrate 1, 3-dichloro-2-propanol (DCP) to (R)- epichlorohydrin (ECH). The second reaction was ring-opening of (R)-ECH to (R)-4-chloro-3- hydroxybutyronitrile (HBN) with cyanide ion (CN-) as a nucleophile. (R)-HBN is a precursor to many valuable chemicals, such as L- carnitine, which plays a vital role in human metabolism. [6, 7] By using mathematical modelling and simulations, optimal reaction conditions were found for this system. Kinetics research has shown that the best results are achieved when both reaction steps are carried out simultaneously in one pot reaching the maximum product yield, and shifting the equilibrium to the right side. High product concentrations cannot be achieved in a batch reactor because of the substrate inhibition. However, in the fed-batch reactor concentrations of the inhibitors can be controlled which makes it an ideal solution. Funding: European Structural and Investment Funds, KK.01.1.1.04. References [1] M. Sudar, Z. Findrik Blažević, Enzyme Cascade Kinetic Modelling, Enzyme Cascade Design and Modelling, editors: S. Kara, F. Rudroff, Springer International Publishing, 2021, 91-108. [2] M. Česnik et al. Chemical Engineering Research and Design, 2019, 150, 140-152. [3] Đ. Vasić Rački et al. Applied Microbiology and Biotechnology, 2011, 91, 845-856. [4] Đ. Vasić Rački et al. Chemical and Biochemical Engineering Quarterly, 2003, 17(1), 3-14. [5] M. Sudar et al. Journal of Biotechnology, 2018, 268, 71-80. [6] T. Suzuki et al. Bioorganic & Medicinal Chemistry Letters, 1996, 6, 2581-2584. [7] Z. Findrik Blažević et al. Advanced Synthesis & Catalysis, 2021, 363(2), 388-410. more...
- Published
- 2021
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