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Stabilisierung Mikrobieller Lipasen

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Stabilisierung Mikrobieller Lipasen Untersuchungen zum Inaktivierungsmechanismus von mikrobiellen Lipasen in wasserfreien Transesterifizierungen Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Benjamin Franken aus Aachen Oktober 2008 Aus dem Institut für Molekulare Enzymtechnologie (IMET) der Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referentin: Frau Prof. Dr. Martina Pohl Korreferent: Herr Prof. Dr. Karl-Erich Jäger Tag der mündlichen Prüfung: 18.11.2008 Die vorliegende Arbeit wurde in der Zeit von Oktober 2005 bis Juni 2008 am Institut für Molekulare Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich unter der Anleitung von Frau Prof. Dr. Martina Pohl und Herrn Prof. Dr. Karl-Erich Jäger angefertigt. Die Arbeit wurde von der Deutschen Forschungsgemeinschaft im Rahmen des Graduiertenkollegs 1166 "Biocatalysis in non-conventional media (BioNoCo)" gefördert. Diese Dissertation wird u.a. in elektronischer Form auf dem WWW-Server der Universitäts- und Landesbibliothek Düsseldorf veröffentlicht: meinen Eltern, Brüdern, Nadine und Freunden. Und so sehen wir betroffen, den Vorhang zu und alle Fragen offen. Marcel Reich-Ranicki (nach einem Zitat von Berthold Brecht aus „Der gute Mensch von Sezuan“) Wissenschaftliche Veröffentlichungen im Rahmen dieser Promotion Artikel in Fachjournalen - Franken, B., Eggert, T., Jaeger, K.-E. and Pohl, M. (2009): Novel insights into the acetaldehyde-induced deactivation mechanism of microbial lipases. Manuskript in Vorbereitung Tagungsbeiträge - Franken, B., Eggert, T., Jaeger, K.-E. and Pohl, M. (2007): Novel strategy to overcome acetaldehyde inactivation of microbial lipases. Vortrag, VAAM-Jahrestagung 2007 (Osnabrück). - Franken, B., Eggert, T., Jaeger, K.-E. and Pohl, M. (2007): New insights into the inactivation mechanism of microbial lipases by acetaldehyde. Posterbeitrag, Biotrans 2007 (Oviedo). - Franken, B., Eggert, T., Jaeger, K.-E. and Pohl, M. (2009): Novel mechanistic insights into the inactivation mechanism of microbial lipases by acetaldehyde. Posterbeitrag, Biotrans 2009 (Bern). In Vorbereitung Danksagung Frau Prof. Dr. Martina Pohl gebührt mehr als der an dieser Stelle übliche Dank. Ich danke ihr für ihre engagierte und freundschaftliche Betreuung, für die stete Diskussionsbereitschaft und die Hilfe bei der Planung und Durchführung dieser Promotionsarbeit sowie für die kritische Durchsicht des Manuskripts. Es war eine große Freude, eine Wissenschaftlerin kennenzulernen, für die der Begriff „Doktormutter“ mehr als nur ein Wort ist. Vielen Dank für die schöne Zeit. Für die freundliche Übernahme des Korreferats, die Hilfe und das stets „offene Ohr“ danke ich Herrn Prof. Dr. Karl-Erich Jäger. Dem DFG-Graduiertenkolleg 1166 BioNoCo „Biocatalysis in non-conventional media“ gilt mein Dank für die Finanzierung meiner Promotionsstelle im Rahmen des mir zuerkannten Stipendiums und die vielen interessanten und lehrreichen wissenschaftlichen und nicht- wissenschaftlichen Veranstaltungen und Kooperationen. Ich bedanke mich bei Herrn PD Dr. Thorsten Eggert für die freundliche Aufnahme in seine Arbeitsgruppe und die engagierte Betreuung in der Anfangsphase meiner Promotion. Ich wünsche ihm und allen Mitarbeitern der evocatal GmbH viel Glück und Erfolg in der Zukunft. Herrn Dr. Sanjib Kumar Karmee (Institut für technische und makromolekulare Chemie der RWTH Aachen, AK Leitner) danke ich für spontane Hilfe bei den GC/MS-Messungen und der Auswertung der Spektren. Frau Dr. Melanie Brocker (Institut für Biotechnologie 1 des Forschungszentrums Jülich, AG Biochemie) und Frau Dr. Sabine Metzger (Biologisch-Medizinisches Forschungszentrum der Heinrich-Heine Universität Düsseldorf, Analytisches Zentrallabor) möchte ich für die spontane, tatkräftige und unermüdliche Unterstützung bei den MALDI-TOF-MS Messungen danken. Allen Mitarbeitern des Instituts für molekulare Enzymtechnologie danke ich für das angenehme und freunschaftliche Arbeitsklima während meiner gesamten Promotionszeit. Ganz besonders herzlicher Dank gilt den Mitarbeitern der Arbeitsgruppen „Gerichtete Evolution“ und „Angewandte Enzymtechnologie“, die mir in jeder Situation mit Rat, Tat und Spaß zur Seite gestanden haben. INHALT 1 Einleitung ............................................................................................................................................. 1 1.1 Enzyme als Katalysatoren .............................................................................................................. 1 1.2 Lipasen ......................................................................................................................................... 3 1.2.1 Allgemeine molekulare und biochemische Eigenschaften ........................................................... 3 1.2.2 Die α/β -Hydrolase-Faltung ........................................................................................................... 6 1.2.3 Lipasen versus Esterasen: Das Konzept der Grenzflächenaktivierung .......................................... 9 1.2.3 Der Katalysemechanismus von Lipasen ...................................................................................... 12 1.3 Hydrolasen in der Biotechnologie ................................................................................................ 14 1.3.1 Lipasen in organischen Lösungsmitteln: Wichtige Konzepte und Anwendungen ...................... 15 1.3.2 Lipase-katalysierte Estersynthese ............................................................................................... 25 1.3.3 Inaktivierung mikrobieller Lipasen durch Acetaldehyd .............................................................. 29 2 Motivation und Zielsetzung ................................................................................................................ 33 2.1 Motivation .................................................................................................................................. 33 2.2 Zielsetzung .................................................................................................................................. 34 3 Material und Methoden ..................................................................................................................... 36 3.1 Material ...................................................................................................................................... 36 3.1.1 Geräte und Chemikalien ............................................................................................................. 36 3.1.2 Bakterienstämme und Vektoren ................................................................................................. 38 3.2 Kultivierung und Stammhaltung von E. coli .................................................................................. 39 3.3 Molekularbiologische Methoden ................................................................................................. 40 3.3.1 Präparation von Plasmid-DNA aus E. coli .................................................................................... 40 3.3.2 QuikChange®-PCR ....................................................................................................................... 40 3.3.3 Hydrolytische Spaltung von DNA durch Restriktionsendonukleasen ......................................... 42 3.3.4 Alkalische Agarosegelelektrophorese von DNA .......................................................................... 42 3.3.5 Hitzeschock-Transformation von E. coli ...................................................................................... 43 3.4 Heterologe Genexpression in E. coli ............................................................................................. 44 3.4.1 Hochzelldichte-Kultivierung ........................................................................................................ 44 3.4.2 Zellaufschluss durch Ultraschall .................................................................................................. 46 3.5 Quantitative Proteinbestimmung ................................................................................................ 47 3.6 Chromatographische Reinigung von BSLB .................................................................................... 48 3.6.1 IMAC an Ni-NTA-Agarose ............................................................................................................ 48 3.6.2 Gelfiltrationschromatographie an Sephadex™ G-25-Medium .................................................... 49 3.7 Lyophilisierung ............................................................................................................................ 49 3.8 Proteinmodifikationen ................................................................................................................ 50 3.8.1 Modifikation mit Acetaldehyd unter nicht-wässrigen Bedingungen .......................................... 50 3.8.2 Modifikation von BSL-B mit Methylacetimidat ........................................................................... 50 3.8.3 Modifikation von BSL-B mit Acetaldehyd unter wässrigen Bedingungen ................................... 51 3.8.4 Modifikation mit 2,4-Hexadienal unter wässrigen Bedingungen ............................................... 51 3.8.5 Modifikation von Rinderserumalbumin
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