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Nebenreaktionen Der Ectoin-Synthase Aus Halomonas Elongata DSM 2581T Und Entwicklung Eines Salzinduzierten Expressionssystems

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Nebenreaktionen Der Ectoin-Synthase Aus Halomonas Elongata DSM 2581T Und Entwicklung Eines Salzinduzierten Expressionssystems Nebenreaktionen der Ectoin-Synthase aus Halomonas elongata DSM 2581T und Entwicklung eines salzinduzierten Expressionssystems Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Elisabeth Witt aus Siegburg Bonn, im Dezember 2011 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. Erwin A. Galinski 2. Gutachterin: apl. Prof. Dr. Christiane Dahl Tag der Promotion: 28.02.2012 Erscheinungsjahr: 2012 Nadelförmige Kristalle der Nγ-Acetyl-diaminobuttersäure (ADABA) Justus von Liebig (1803-1873) Rosarium Philosophorum, 1550 H. elongata, mikroskopiert in der stationären Phase, demonstriert eindrucksvoll den namensgebenden Pleomorphismus Teile dieser Arbeit wurden im Rahmen von Tagungs- und Zeitschriftenbeiträgen publiziert. Poster Witt E, Stein M, Kurz M, Galinski EA (2005) A substitute compatible solute of the ectoine deletion mutant Halomonas elongata KB1. VAAM-Jahrestagung Göttingen; EXP025 Witt E, Ures A, Stein M, Galinski EA (2007) Production and purification of N-γ-acetyl-L-2,4- diaminobutyric acid, the precursor of the compatible solute ectoine. VAAM-Jahrestagung Osnabrück; PB056 Stein M, Ures A, Witt E, Schwarz T, Galinski EA (2008) Whole-cell biocatalysis for the stereospecific hydroxylation of cyclic compatible solutes. VAAM-Jahrestagung Frankfurt; PC20 Witt E, Galinski EA (2010) Ectoine-Synthase: Substrate Spectrum and Reversibility. VAAM- Jahrestagung Hannover; BTP44 Korsten A, Witt E, Galinski EA (2010) Isolation and characterization of the unusual compatible solute NAGGN. VAAM-Jahrestagung Hannover; BTP43 Witt E, Grün A, Kurz M, Galinski EA (2011) Engineered salt-induced ectoine promoter for use in H. elongata as halophilic expression system. VAAM-Jahrestagung Karlsruhe, GWP029 (ausgezeichnet mit dem Poster-Preis) Vorträge Witt E, Stein M, Galinski EA (2007) Ectoine-synthase: action and side reaction. Halophiles Essex, Colchester, UK; O42 Witt EMHJ, Galinski EA (2010) Unexpected property of ectoine-synthase and its application for the production of the novel compatible solute ADPC. Halophiles Beijing, China; O31 Witt E, Galinski EA (2010) Improvement of salt-induced ectoine promoter for heterologous expression in H. elongata. Osmomeeting, Marburg Zeitschriftenartikel Witt EMHJ, Davies NW, Galinski EA (2011) Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC. Appl Microbiol Biotechnol 91 (1): 113-122 Inhaltsverzeichnis Verzeichnis der Abbildungen und Tabellen ................................................................................................................. VIII Verzeichnis der Abkürzungen und Trivialnamen ........................................................................................................ XI I Einleitung ................................................................................................................................................................................ 1 1 Extremophile Organismen ................................................................................................... 1 2 Halophile Mikroorganismen ................................................................................................ 2 2.1 Lebensräume und Problematik der hyperosmolaren Lebensräume ............................ 3 2.2 Strategie der Anpassung halophiler Organismen ......................................................... 4 2.2.1 „Salt-in-cytoplasm“-Strategie ................................................................................ 4 2.2.2 „Compatible-solute“-Strategie .............................................................................. 5 3 Kompatible Solute ............................................................................................................... 6 3.1 Wirkweise ...................................................................................................................... 7 3.2 Stabilisierung von Biomolekülen ................................................................................... 8 3.3 Solute in der Industrie ................................................................................................... 9 3.3.1 Produktion ............................................................................................................. 9 3.3.2 Anwendungen ........................................................................................................ 9 3.4 Neue Solute / Derivate bekannter Solute ...................................................................10 4 Halomonas elongata .........................................................................................................11 4.1 Ectoinbiosynthese in H. elongata ...............................................................................12 4.1.1 Die Entdeckung des ADPC ....................................................................................14 5 Ziel der Arbeit ....................................................................................................................15 II Material und Methoden ................................................................................................................................................. 17 1 Verwendete Bakterienstämme und Plasmide ...................................................................17 2 Nährmedien .......................................................................................................................19 2.1 Medien für Flüssigkultur und Stammhaltung .............................................................19 2.2 Medien für die Hochzelldichte-Fermentation.............................................................20 2.3 Medien für Molekularbiologie und Proteinbiochemie ...............................................21 2.4 Medienzusätze und Supplementierungen ..................................................................22 2.4.1 Antibiotika ............................................................................................................22 2.4.2 Supplemente ........................................................................................................23 2.5 Agarplatten ..................................................................................................................23 3 Puffer und Lösungen..........................................................................................................23 3.1 Puffer und Lösungen für die Analytik ..........................................................................23 Inhaltsverzeichnis 3.2 Puffer und Lösungen für die Molekularbiologie ........................................................ 24 3.3 Puffer und Lösungen für die Proteinbiochemie ......................................................... 24 4 Kultivierungsverfahren ..................................................................................................... 26 4.1 Stammhaltung von Bakterien ..................................................................................... 26 4.2 Bakterienanzucht........................................................................................................ 26 4.2.1 Schüttelkolben .................................................................................................... 26 4.2.2 Mikrotiterplatte .................................................................................................. 26 4.2.3 Fermenter ........................................................................................................... 27 4.2.3.1 Fermentation im 5 L-Maßstab ....................................................................... 27 4.2.3.2 Fermentation in 15 L-Fed-Batch-Verfahren ................................................... 28 4.3 Verfolgung des Zellwachstums ................................................................................... 29 4.4 Ernte ........................................................................................................................... 29 5 Analytik ............................................................................................................................. 29 5.1 Gefriertrocknung ........................................................................................................ 29 5.2 Isokratische HPLC ....................................................................................................... 29 5.2.1 Probenaufbereitung ............................................................................................ 30 5.2.2 HPLC-Messung .................................................................................................... 31 5.3 FMOC-ADAM-HPLC ..................................................................................................... 31 5.3.1 Probenaufbereitung ............................................................................................ 32 5.3.2 FMOC-ADAM-HPLC-Messung ............................................................................. 32 5.4 13C-NMR ...................................................................................................................... 33 5.4.1 Probenaufbereitung ............................................................................................ 34 5.4.2 NMR-Messung ..................................................................................................... 34 5.5 Glucose-Test ..............................................................................................................
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