Zeuner, Birgitte

Afhandlingen undersøger forskellige metoder til at øge reaktionsudbyttet i enzymkatalyserede syntesereaktioner. Først undersøges brugen af ukonventionelle reaktionsmedier som ioniske væsker og organiske solventer, både som hovedsolvent og co-solvent. Til sidst undersøges brugen af mere klassiske metoder i reaktionsdesign såsom enzymimmobilisering og brug af en membranreaktor. To forskellige enzymtyper anvendes, nemlig ferulinsyreesteraser og sialidaser.Med sinapoylering af glycerol som eksempel blev det vist, at både anionen i den ioniske væske og ferulinsyreesterasernes struktur havde betydning for ferulinsyreesteraseaktivitet og -stabilitet i ionisk væske-systemer med 15 % (v/v) vand (buffer). Den kvantekemibaserede software COSMO-RS kunne forklare den observerede anioneffekt som et resultat af anionens tendens til at danne hydrogenbindinger med enzymstrukturen. Anvendeligheden af COSMO-RS og andre termodynamiske modeller i forbindelse med valg af solventer til ferulinsyreesterasekatalyserede reaktioner blev undersøgt. Ferulinsyreesterase type A fra Aspergillus niger og ferulinsyreesterasene i den kommercielle enzympreparation fra Humicola insolens, Depol 740L, kunne ikke katalysere esterificeringen af arabinose eller xylose med hydroxycinnamater i systemer af ionisk væske og buffer eller i mikroemulsioner. Dog kunne begge ferulinsyreesteraser katalysere feruloylering og/eller sinapoylering af kationen C2OHMIm+; dette understreger at ferulinsyreesteraserne har en bred acceptorspecificitet og således har potentiale for at kunne gøre nytte i andre, mere brugbare reaktioner med solventet.En modificeret sialidase fra Trypanosoma rangeli, Tr6, kan katalysere trans-sialylering, fx produktion af 3’-sialyllaktose fra laktose og kaseinglykomakropeptid (CGMP), men reaktionsudbyttet trækkes ned af substrat- og produkthydrolyse. Ved at tilsætte 20-25 % (v/v) t-butanol blev trans-sialyleringsudbyttet øget 1,4 gange, og forholdet mellem syntese og hydrolyse blev øget 1,2 gange. Forholdet mellem syntese og hydrolyse blev også øget ved at tilsætte ioniske væsker som co-solventer, men på bekostning af enzymaktiviteten. Tilbage i vandigt miljø kunne immobilisering af Tr6 på magnetiske nanopartikler øge forholdet mellem syntese og hydrolyse 2,1 gange og desuden give en 2,5 gange forøget biokatalytisk produktivitet (mg produkt pr. mg enzym). Desværre tabte de magnetiske nanopartikler en stor del af enzymaktiviteten i brug og kunne kun genbruges få gange. I stedet kunne frit Tr6 bruges i en membranreaktor med et 1,3 gange højere udbytte på sialyldonoren (CGMP) til følge; dette understreger, at kontinuert fjernelse af produktet vha. en membranreaktor er en brugbar metode til at undgå produkthydrolyse. Stabiliteten af Tr6 var høj i membranreaktoren, og 7 ganges genbrug resulterede i mere end 9 ganges forøgelse af den biokatalytiske produktivitet.Det kan konkluderes, at omhyggelig udvælgelse af både enzymstrukturer og solventsystemer nødvendig i reaktioner hvor ukonventionelle solventer kræves for at opnå et tilfredsstillende vi reaktionsudbytte, fx i den termodynamisk kontrollerede esterificering katalyseret af ferulinsyreesteraser. Med den modificerede sialidase Tr6 var resultaterne derimod mest lovende, når katalysen fandt sted i vandigt reaktionsmedium hjulpet på vej af kontinuert fjernelse af produktet eller enzymimmobilisering.This thesis investigates different methods for improving reaction yields of enzyme-catalysed synthesis reactions. These methods include the use of non-conventional media such as ionic liquids (ILs) and organic solvents as main solvents or as co-solvents as well as the use of more classical reaction design methods, i.e. enzyme immobilization and the use of an enzymatic membrane reactor. Two different enzyme classes, namely feruloyl esterases (FAEs) and sialidases are employed.Using sinapoylation of glycerol as a model reaction it was shown that both the IL anion nature and the FAE structure were important for FAE activity and stability in IL-buffer (15% v/v) systems. The quantum chemistry-based COSMO-RS method was applied for explaining the IL anion effect in terms of hydrogen bonding capacity. Furthermore, the usefulness of COSMO-RS and other thermodynamically based tools in solvent selection for FAE-catalysed acylation reactions was reviewed. FAE type A from Aspergillus niger and an FAE from a commercial preparation from Humicola insolens, Depol 740L, could not catalyse the esterification of arabinose or xylose with hydroxycinnamates in IL-buffer systems or in surfactantless microemulsion. However, both FAEs catalysed the feruloylation and/or sinapoylation of solvent cation C2OHMIm+, thus underlining the broad acceptor specificity of FAEs and their potential for future solvent reactions.An engineered sialidase from Trypanosoma rangeli, Tr6, catalyses trans-sialylation but the yield is hampered by substrate and product hydrolysis. The formation of 3’-sialyllactose from lactose and casein glycomacropeptide was used as a model reaction. Addition of 20-25% (v/v) t-butanol improved the trans-sialylation yield 1.4-fold and the synthesis/hydrolysis ratio 1.2-fold. Using ILs as co-solvents, the synthesis/hydrolysis ratio was also improved, but the trans-sialylation yield decreased, probably due to destabilization of Tr6 caused by the ILs. Returning to the conventional aqueous medium, immobilization of Tr6 on magnetic nanoparticles improved the synthesis/hydrolysis ratio 2.1-fold and increased the biocatalytic productivity of 2.5-fold. However, the recyclability of the immobilized enzyme was low. Reusing Tr6 seven times in a membrane reactor increased the trans-sialylation yield on the limiting substrate 1.3-fold, emphasizing the importance of the continuous product removal. Furthermore, the biocatalytic productivity was increased more than 9-fold as a result of the enzyme recovery.In conclusion, where the use of non-conventional media is required for catalysis, e.g. in the thermodynamically controlled FAE-catalysed esterification, careful selection of both solvent system and the FAE itself is required to obtain adequate reaction yields. In contrast, for Tr6 the most promising results were obtained when keeping the reaction in aqueous medium and employing other reaction design methods such as continuous product removal and enzyme immobilization

Online Research Database In Technology

Solvent engineering and other reaction design methods for favouring enzyme-catalysed synthesis

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Solvent engineering and other reaction design methods for favouring enzyme-catalysed synthesis

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