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Aqueous two-phase extraction of proteins and enzymes using tetraalkylammonium-based ionic liquids
| Content Provider | Semantic Scholar |
|---|---|
| Author | Dreyer, Susanne |
| Copyright Year | 2009 |
| Abstract | To meet the challenges of a growing market of industrial biotechnology, the development of new and innovative downstream methods for proteins and enzymes is of great importance. Within the scope of this thesis, the application of ionic liquids for the generation of aqueous two-phase systems and their potential use as an alternative technique for the purification of catalytically active enzymes was evaluated. The first objective comprised the characterisation of ionic liquid-based aqueous two-phase systems (IL-based ATPS). Several factors influencing the phase formation and composition were investigated, such as the type of phase-forming compounds, temperature and pH. By studying the partitioning of different model proteins, correlations between the protein features and system properties could be found, thereby providing a deeper understanding of the mechanisms influencing the partition process. Based on these findings, electrostatic interaction has been identified as the main driving force of protein partitioning in IL-based ATPS. Moreover, a statistical model based on the model proteins’ physico-chemical properties has been established and provides a good start for predicting the protein partitioning in IL-based ATPS. With regard to the purification of enzymes, the main obstacle in developing a suitable extraction method represents the maintenance of the stability and the catalytic activity of the biocatalyst. Therefore, the application of IL-based ATPS for biocompatible extraction of enzymes from complex mixtures such as cell lysates was investigated. It was demonstrated that the ionic liquid Ammoeng 110 in combination with an inorganic salt (K2HPO4/KH2PO4) can be used to establish an IL-based ATPS which provides the necessary conditions of pH and biocompatibility in order to maintain enzyme activity. Using efficient experimental design the partitioning of an ADH from Lactobacillus brevis (LB ADH) and an ADH from a thermophilic organism (T ADH) from a cell extract of E. coli was optimised with respect to the enrichment of the enzyme in the IL-containing phase. The specific enzyme activity in the upper phase could be increased to 206.7 ± 5.3 % for LB ADH and 403.4 ± 5.4 % for T ADH. The presence of ionic liquid within IL-based ATPS was also found to be advantageous thanks to its stability enhancing effect on both enzymes and its solubility enhancing effect on hydrophobic substrates. Thus, it could be shown that the IL-based ATPS offers the opportunity to combine the purification process of active biocatalysts with the performance of enzyme-catalysed reactions especially in case of the conversion of hydrophobic substrates. Advantages such as improved conversion and yield could be observed. Parts of this thesis as well as further results not included in this work have already been presented, published and filed as a patent. Publications Dreyer S, Lembrecht J, Schumacher J, Kragl U. (2007). Enzyme catalysis in non-aqueous media past-present-future. In R. Patel (Ed.) Biocatalysis in the Pharmaceutical and Biotechnology Industries, Taylor & Francis Group, Chapter 33, 791-829 Klembt S, Dreyer S, Eckstein M, Kragl U. (2007). Biocatalytic reactions in ionic liquids. In Wasserscheid, P., Welton, T. (eds.): Ionic Liquids in Synthesis (2 edition), Wiley-VCH Weinheim Dreyer S, Kragl U. (2008). Ionic liquids for aqueous two phase extraction and stabilization of enzymes. Biotechnology and Bioengineering, Vol. 99, 6, 1416-1424 Dreyer S, Salim P, Kragl U. (2008). Driving forces of protein partitioning in an ionic liquidbased aqueous two phase system. Submitted to Biochemical Engineering Journal Patents Dreyer S, Kragl U. (2007). Verfahren zur Extraktion von Biomolekulen (Process for the extraction of biomolecules). German patent application, 102007001347.9, 04/01/2007 Altrichter J, Kragl U, Dreyer S (2008). Stabilisierung von Zellen durch ionische Flussigkeiten (Stabilisation of cells by ionic liquids). German patent application, 102008039734.2, 26/08/2008 Oral presentations Dreyer S. (19/06/2007). Application of ionic liquids for aqueous two phase extraction. International Conference on Biopartitioning and Purification, Lisbon Dreyer S. (16/11/2007). Ionic liquids for the purification and stabilisation of proteins. Workshop Interaction of Ionic Liquids and Proteins, Rostock Dreyer S. (21/02/2008). Use of ionic liquids for protein purification. SPP1191 Ionic Liquid Winterschool, Leipzig Dreyer S. (03/09/2008). Ionic liquids for aqueous two phase extraction of enzymes: application and understanding. Presented by Julia Duwensee, Biocat, Hamburg Poster presentations Dreyer S, Trapp S, Kragl U. (30/05 01/06/2007). Stabilisation of two different alcohol dehydrogenases by a readily available ionic liquid. European Bioperspectives, Koln Dreyer S, Kragl U. (05/08 10/08/2007). Ionic liquid-based aqueous two phase extraction of enzymes. 2 International Congress on Ionic Liquids (COIL 2), Yokohama Dreyer S, Kragl U. (09/10 11/10/2007). Ionische Flussigkeiten fur die Extraktion und Stabilisierung von Proteinen. Biotechnica, Hannover Dreyer S, Kragl U. (15/11 16/11/2007). Ionic liquid-based aqueous two phase extraction of enzymes. Workshop Interaction of Ionic Liquids and Proteins, Rostock Dreyer S, Kragl U. (27/03 29/03/2008). Protein distribution in ionic liquid-based aqueous two phase systems. International 10 Fruhjahrssymposium of GDCh JCF, Rostock |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://rosdok.uni-rostock.de/file/rosdok_derivate_000000003754/Dissertation_Dreyer_2009.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
