Biotechnological production; protein engineering
Final Report Abstract
Based on the first phase of project, the cultivation of E. coli K1 in a 10 l bioreactor was optimized. For this, the polySia production and chain length was studied by using design of experiments in the Batch process. It is shown that the polySia production and chain length are dependent on different parameters. The investigation of the fed-batch cultivation on productivity and chain length was carried out using different growth rates during the fed-batch-phase. Afterwards, a new purification strategy based on membrane adsorber technology and inorganic clay minerals was developed. This enabled the yields to be increased significantly compared to the precipitation from the first phase of the project. The yield of the new purification strategy was 61% for the batch and 40% for the fed-batch cultivation. Chain lengths over 145 DP have been isolated in the fed-batch cultivation. The purity of polySia is 97-99% after purification. The purified polySia were provided to project partners. A variety of inactive forms of endoN have been produced and have been successfully used in collaborative studies to stain histological sections and to identify novel polySia carriers (ongoing study in collaboration with Prof. Michael Sixt, IST Austria).
Publications
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Produktion und Aufarbeitung von Polysialinsäure aus E. coli K1, 27. Dechema-Jahrestagung der Biotechnologen 2009, Mannheim, Germany, 08.- 10.09.2009
Bice I.; Rode B., Beutel, S. und Scheper, T.
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Proteolytic Release of the Intramolecular Chaperone Domain Confers Processivity to Endosialidase F. 8th Carbohydrate Bioengineering Meeting, Ischia, Napels, Italy,10.-15.05.2009
Schwarzer D, Stummeyer K, Haselhorst T, Freiberger F, Rode B, Scheper T, von Itzstein M, Mühlenhoff M, Gerardy-Schahn R
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Aufreinigung von Polysialinsäure mithilfe von Adsorbern, Chemie Ingenieur Technik 82 (2010), 1497. Dechema-Jahrestagung der Biotechnologen, Aachen, Germany, 21.-23.09.2010
Bice, I.; Celik, H.; Beutel S. und Scheper, T.
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Leiman Petr. Crystal Structure of Coliphage Phi92 Endosialidase - A New Family Member with a Different Substrate Specficity. Viruses of Microbes, Paris, France, June 21-25, 2010
Schwarzer D, Stummeyer K, Bethe A, Oberbeck A, Mühlenhoff M, Gerardy-Schahn R, Leiman P
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(2011). Comparison of polysialic acid production in Escherichia coli K1 during batch cultivation and fed-batch cultivation applying two different control strategies. J. Biotechnol., 154, 222-229
Chen,R., John,J., Rode,B., Hitzmann,B., Gerardy-Schahn,R., Kasper,C., and Scheper,T.
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Chain length determination of polysialic acid isolated from E. coli K1batch and fed- batch cultivations, 1st European Congress of Applied Biotechnology 2011, Berlin, Germany, 25.-29.09.2011
Bice, I.; Solle, D.; Beutel, S. and Scheper T.
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Optimisation of polysialic acid purification by using adsorber materials, 1st European Congress of Applied Biotechnology 2011, Berlin, Germany, 25.- 29.09.2011
Celik, H.; Bice, I.; Beutel, S. and Scheper, T.
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(2014). Downstream processing of high chain length polysialic acid using membrane adsorbers and clay minerals for application in tissue engineering. Engineering in Life Sciences, 13, 140-148
Bice,I., Celik,H., Wolff,C., Beutel,S., Zahid,M., Hitzmann,B., Rinas,U., Kasper, C., Gerardy- Schahn,R., and Scheper,T.
