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

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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.
Original languageEnglish
PublisherTechnical University of Denmark, Department of Chemical and Biochemical Engineering
Number of pages195
Publication statusPublished - 2014


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