20.500.12556/RUL-140014 Vpliv dodatka kofaktorja na aktivnost amin transaminaze imobilizirane v obliki zamreženih encimskih agregatov Effect of cofactor addition on the activity of amine transaminase immobilised as crosslinked enzyme aggregates Biokataliza igra pomembno vlogo v prehodu k trajnostnim načinom proizvodnje. Amin transaminaze (ATA) omogočajo učinkovito stereoselektivno proizvodnjo aminov in so zato dobra alternativa kemijski sintezi pri pridobivanju optično čistih spojin. Implementacijo ATA v industrijsko proizvodnjo ovira slaba stabilnost encimov, nizka zadržana aktivnost po imobilizaciji ter potreba po eksogeno dodanem kofaktorju piridoksal-5'-fosfatu (PLP). V zadnjih letih je proteinski inženiring omogočil izražanje izjemno stabilnih amin transaminaz z visoko afiniteto za kofaktor. Poleg tega priprava zamreženih encimskih agregatov (CLEA) proizvedenih v mikropretočnih sistemih in njihova uporaba v mikroreaktorjih ponujata obetaven način za dolgotrajno uporabo encimov v kontinuirnih biokatalitskih procesih. V pričujočem delu smo raziskali vpliv eksogenega dodatka kofaktorja PLP na zadržano aktivnost in stabilnost CLEA delcev proizvedenih z izbrano ATA. Encimskim agregatom pripravljenim z mikropretočno napravo smo določali velikost na osnovi merjenja dinamičnega sipanja svetlobe in elektronskega mikroskopiranja. Raziskali smo aktivnost in stabilnost pripravljenih CLEA delcev v kontinuirnem biokatalitskem procesu v membranskem mikroreaktorju z in brez eksogenega PLP v napajalnem toku. Rezultati kažejo na visoko afiniteto izbrane amin transaminaze za kofaktor PLP in visoke zadržane aktivnosti CLEA delcev tega encima tudi v odsotnosti eksogeno dodanega kofaktorja. To smo potrdili tudi v procesu izvedenem v membranskem mikroreaktorju z imobiliziranimi CLEA delci, kjer ni bilo bistvenih razlik v konverziji reaktanta na izhodu iz mikroreaktorja, ko je ta obratoval s PLP ali brez njega v napajalnem toku. Priprava CLEA delcev v mikropretočnem sistemu se je izkazala kot zanesljiva in učinkovita metoda za imobilizacijo ter implementacijo ATA v industrijski biokatalizi. Biocatalysis is playing a major role in the transition toward sustainable production. Transaminases are enzymes that enable the stereoselective production of amines and are a good alternative to traditional chemical synthesis and chiral resolution. In the past wider implementation of amine transaminases in industry has been hampered by poor operational stability, low recovered activity of immobilised enzyme and the need to add the cofactor pyridoxal-5'-phosphate to the reaction mixture. In recent years protein engineering has enabled the expression of highly stable transaminases with exceptional affinity for the cofactor. Furthermore, microfluidics-based crosslinked enzyme aggregate preparation offers a promising way of using enzymes in continuous biocatalytic processes. In the present work we look at the effect of exogenous PLP addition on the recovered activity and stability of CLEA particles prepared with the selected ATA. We determined the size and shape of microfluidicaly prepared particles with dynamic light scattering and electron microscopy. We also measured the activity and stability of the prepared CLEA particles in a continuous biocatalytic process in a membrane microreactor, with and without exogenous PLP in the feed flow. The results show the chosen amine transaminase exhibits a high affinity for the cofactor PLP, and high recovered activities of CLEA particles prepared with the enzyme even in absence of exogenously added cofactor. We also confirmed this in the membrane microreactor process with immobilised CLEA particles, where we saw no significant differences in reactant conversion in the microreactor eluate, while it was operating with or without exogenous PLP in the reactor feed. CLEA amin transaminaza piridoksal-5'-fosfat mikroreaktor biokataliza CLEA amine transaminase pyridoxal-5'-phosphate microreactor biocatalysis true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2022-09-09 12:40:00 2022-09-09 12:40:07 2022-11-17 14:04:46 0000-00-00 00:00:00 2022 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 21784 129822467 Hlebanja_Patrik_-_Vpliv_dodatka_kofaktorja_na_aktivnost_amin_transaminaze_imobilizirane_v_obliki.pdf Hlebanja_Patrik_-_Vpliv_dodatka_kofaktorja_na_aktivnost_amin_transaminaze_imobilizirane_v_obliki.pdf 1 F7E1E58A73240F01C6357D83FE298A9B 4e7092212a5d31d9c5f547efa1fd00fda73ec8e134e660b848a81fc83df4bee9 b78176f1-302b-11ed-92af-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=160672 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0