Enzymkatalysierte Synthese chiraler Substanzen unter hohen Drücken
Zusammenfassung der Projektergebnisse
The main focus of the first part of the project was online monitoring of C-C bond formations. This study is a first-time application of 2D-fluorescence spectroscopy for the online monitoring of 2-hydroxy ketone synthesis which is of a high interest in pharmaceutical industry. So far examples for the use of IR spectroscopy to monitor biotransformations have demonstrated its high potential for precise analyses as well as its flexible instrumentation, which both afford a wide range of applications. Despite its advantages the cost of IR devices is still a negative aspect. Thus, here, 2D-fluorescence spectroscopy could be attractive for industry because of its low cost. Moreover, we showed that BFD-catalysed reactions starting with 20 - 40 mM benzaldehyde and 400 mM acetaldehyde could be successfully followed although this concentration range has not been used in fluorescence measurements so far. Here, online monitoring offers not only the ease of getting data without time-consuming sample preparation for offline analysis but also precise determination of kinetic characteristics of the enzyme. Furthermore, 2D-fluorescence spectroscopy was applied for the monitoring of carboligations catalysed at 100 MPa. Although it was well known that fluorescence spectroscopy is quite sensitive to any kind of variations in the physical and chemical properties of reaction sample analysed, the passion of this investigation was to follow C-C bond formations which was the first-time monitoring of these reactions catalysed at relatively extreme conditions. Novozym 435 was applied for the aminolysis of rac-3 and 1 towards the enantioselective synthesis of the β-amino acid ester (S)-3 via kinetic resolution. Reactions were conducted in the solvents THF and DIPE at high pressure and at ambient pressure. The enantioselectivity in THF remained unchanged at ambient pressure or 200 MPa. A decreased enantioselectivity probably resulting from conformational changes was observed at 200 MPa as compared to ambient pressure in DIPE as a solvent. Reactions were slowed down under pressure in both solvents hinting at positive activation volumes for the reaction. A similar stability was observed in THF at ambient and high pressure. In combination with the finding of a reduced reaction rate, reversible changes in the protein structure are anticipated. Because of pulverization of the carrier material in DIPE both at ambient and high pressure, no reliable information on enzyme stability could be gained in the respective solvent. The investigation of the influence of solvent composition and temperature on the activity and enantioselectivity of the CRL catalyzed transesterification show that most of the effects which can be seen by varying the reaction medium or the reaction temperature are due to motion and interaction of water molecules on and with the enzyme surface. The results of the experiments with binary mixtures of hexane and tetrahydrofurane indicate that the decrease in conversion and the increase in selectivity with increasing solvent hydrophilicity are induced by different water contents on the enzyme surface and not by the solvent itself. This observation is supported by results from catalysis at different reaction temperatures. It could be shown, that the decrease in conversion and the increase in selectivity at higher temperatures only takes place in reaction media with hydrophobic solvent parts. In this case the stripping effect is enlarged with higher temperatures. Neither conversion, nor enantiomeric excess are influenced by the reaction temperature in the hydrophilic solvent, which verify the conclusion that enantioselectivity is mainly influenced by the water content on the enzyme surface and not by the solvent itself. For our knowledge, this relationship is shown for the first time in such a clear way. Furthermore the investigation of the pressure effect on the lipase catalyzed transesterification in organic solvents gives a deeper insight in the mechanisms of intra- and intermolecular interactions. In organic solvents the preservation of the enzyme activity is attributed mainly lo the presence of enzyme-bound water. High pressures favor the hydration of polar groups and thereby decrease stripping effects, which occur in slightly hydrophilic organic solvents. The observed enlargement of activity with increasing pressure is possibly due to the preservation of the enzyme bond water. This observation confirms the model that water acts like a „lubricant" that weakens intermolecular interactions between single amino acids of the protein and thereby preserves the fiexibility and the activity of the enzyme respectively.
Projektbezogene Publikationen (Auswahl)
- "Fluorescence spectroscopy as a novel method for online analysis of biocatalytic C-C bond formations" Journal of Molecular Catalysis B: Enzymatic 2010; 66 (1-2): 124 - 129
Kara S, Anton F, Solle D, Neumann M, Hitzmann B, Scheper T, Liese A
- "Immobilisation and characterisation of benzoylformate decarboxylase from Pseudomonas putida on spherical silica" Bioprocess and Biosystems Engineering 2011; 34 (6): 671 - 680
Peper S, Kara S, Long WS, Liese A, Niemeyer B
- "Influence of reaction conditions on the enantioselectivity of biocatalysed C-C bond formations under high pressure conditions" Journal of Biotechnology 2011; 152(3): 87 - 92
Kara S, Long WS, Berheide M, Peper M, Niemeyer B, Liese A
- "Online analysis methods for monitoring of bioprocesses" chimicaoggi - Chemistry TODAY 2011; 29 (2): 38 - 4 1
Kara S, Müller JJ, Liese A
- PhD Thesis: "Online monitoring of biocatalytic 2-hydroxy ketone synthesis under ambient and high pressure" Technische Universität Hamburg-Harburg/Hamburg University of Technology, Institute of Technical Biocatalysis, Hamburg, Germany, 2011
Kara S
- "Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification" Journal of Biotechnology 2012
Herbst D, Peper S, Niemeyer B
(Siehe online unter https://doi.org/10.1016/j.jbiotec.2012.03.011)
