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Hydroxynitril-Lyasen Für Die Biotechnologie

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Hydroxynitril-Lyasen Für Die Biotechnologie Hydroxynitril-Lyasen für die Biotechnologie Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Jan-Karl Guterl Aus Essen Jülich, Oktober 2008 Aus dem Institut für Molekulare Enzymtechnologie der Heinrich-Heine Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Frau PD Dr. Martina Pohl Korreferent: Herr Prof. Dr. Michael Bott Tag der mündlichen Prüfung: 27.11.2008 II Die vorliegende Arbeit wurde in der Zeit von Februar 2005 bis Oktober 2008 am Institut für Molekulare Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf im Forschungs- zentrum Jülich unter der Leitung von Frau PD Dr. Martina Pohl angefertigt. Die Arbeit wurde vom Bundesministerium für Bildung und Forschung im Rahmen des Pro- jektes „Biokatalytische Hydrocyanierung und Hydroformylierung (BioHydroForm)“ sowie von der DFG im Rahmen des Graduiertenkollegs „Biocatalysis using non-conventional media (BioNoCo)“ gefördert. III Es könnt alles so einfach sein – isses aber nicht! (Die Fantastischen Vier, „Einfach sein“) IV Wissenschaftliche Veröffentlichungen im Rahmen dieser Promotion Artikel in Fachjournalen: „A high-troughput screening assay for hydroxynitrile lyase activity” Andexer, J.; Guterl, J.-K.; Pohl, M.; Eggert, T. Chemical Communications 40: 4201 – 4203 (2006) „Hydroxynitrile lyase catalyzed cyanohydrin synthesis at high pH-values” von Langermann, J.; Guterl, J.-K.; Pohl, M.; Wajant, H.; Kragl, U. Bioprocess and Biosystems Engineering 31 (3): 155 – 161 (2008) „Uneven twins: comparison of the two enantiocomplementary hydroxynitrile lyases with /- hydrolase fold” Guterl, J.-K.; Andexer, J.; Sehl, T.; von Langermann, J.; Frindi-Wosch, I.; Rosenkranz, T.; Fitter, J.; Gruber, K.; Kragl, U.; Eggert, T.; Pohl, M. Zur Veröffentlichung eingereicht. „The hydroxynitrile lyase from flax – a lyase that looks like an alcohol dehydrogenase” Horeis, G.; Guterl, J.-K.; Pohl, M.; Pichler, S.; Wagner, U.; Gruber, K.; Kratky, C. In Vorbereitung. Tagungsbeiträge: „Directed evolution of a plant hydroxynitrile lyase for industrial applications” Andexer, J.; Guterl, J.-K.; Pohl, M.; Eggert, T. Posterbeitrag, Industrial Biocatalysis in Pharmacy and Fine Chemistry (Nimes/ Frankreich, 2005). „High-throughput screening assay for hydroxynitrile lyases” Andexer, J.; Guterl, J.-K.; Pohl, M.; Eggert, T. Posterbeitrag, VAAM-Tagung (Jena, 2006) V „Improving the hydroxynitrile lyase from Manihot esculenta by directed evolution for techni- cal applications” Guterl, J.-K.; Andexer, J.; Eggert, T.; Pohl, M. Posterbeitrag, Biocat (Hamburg, 2006). „First steps towards the optimization of the hydroxynitrile lyase from Linum usitatissimum” Guterl, J.-K.; Horeis, G.; Gruber, K.; Kratky, C.; Andexer, J.; Eggert, T.; Pohl, M. Posterbeitrag, Biotrans (Oviedo/ Spanien, 2007). VI Inhalt Inhalt Wissenschaftliche Veröffentlichungen im Rahmen dieser Promotion ..................................... V Inhalt........................................................................................................................................VII Abkürzungen ............................................................................................................................XI Verzeichnis der Abbildungen & Tabellen............................................................................. XIV 1. Einleitung ............................................................................................................................... 1 1.1 Die Anwendung von Enzymen in der Biotechnologie..................................................... 1 1.2 Die Hydroxynitril-Lyasen ................................................................................................ 2 1.2.1 Durch Hydroxynitril-Lyasen katalysierte Reaktionen: Cyanogenese & Hydrocyanierung................................................................................................................ 2 1.2.1.1 Die Cyanogenese als Abwehrstrategie höherer Pflanzen....................................... 2 1.2.1.2 Metabolismus der cyanogenen Glycoside.............................................................. 3 1.2.2 HNLs sind durch konvergente Evolution entstanden................................................ 6 1.2.2.1 FAD-abhängige HNLs: Struktur, Substratspektrum und Verbreitung................... 8 1.2.2.2 FAD-unabhängige HNLs: ein kurzer Überblick .................................................. 10 1.2.3 Die HNL aus Manihot esculenta: eine S-selektive /-Hydrolase......................... 11 1.2.4 Die HNL aus Arabidopsis thaliana – Der ungleiche Zwilling der MeHNL ........... 13 1.2.5 Die HNL aus Linum usitatissimum ähnelt Zn2+-abhängigen Alkoholdehydrogenasen................................................................................................... 14 1.2.6 Biotechnologische Nutzung der HNLs ................................................................... 16 1.2.7 Cyanhydrine sind vielseitige Bausteine in der organischen Synthese .................... 17 1.2.8 Randbedingungen für die technische Herstellung enantiomerenreiner Cyanhydrine ..................................................................................................................... 18 1.3 Stabilität von Enzymen .................................................................................................. 20 1.3.1 Allgemeine Aspekte der Stabilität von Enzymen ................................................... 20 1.3.2 Kinetische und Thermodynamische Stabilität......................................................... 21 1.3.3 Allgemeine Aspekte der Inaktivierung von Enzymen ............................................ 23 1.3.4 Einfluss der Temperatur auf die Enzymstabilität.................................................... 24 1.3.5 Einfluss des pH-Wertes auf die Enzymstabilität..................................................... 25 1.3.6 Möglichkeiten zur Stabilisierung von Enzymen ..................................................... 27 1.3.6.1 Rationales Protein-Design.................................................................................... 28 1.3.6.2 Gerichtete Evolution und semi-rationale Ansätze................................................ 29 1.3.6.3 Stabilisierung durch chemische Additive............................................................. 33 1.3.6.4 Stabilisierung durch Immobilisierung.................................................................. 34 1.4 Zielsetzung dieser Arbeit ............................................................................................... 36 2. Material & Methoden........................................................................................................... 38 2.1 Material .......................................................................................................................... 38 2.1.1 Chemikalien & Enzyme .......................................................................................... 38 2.1.2 Geräte & Software................................................................................................... 38 2.1.3 Verwendete Bakterienstämme................................................................................. 40 2.1.4 Verwendete Plasmide.............................................................................................. 41 2.1.5 Oligonukleotide....................................................................................................... 41 2.2 Kultivierung und Lagerung von Bakterien..................................................................... 42 2.2.1 Nährmedien ............................................................................................................. 42 2.2.2 Antibiotika............................................................................................................... 44 2.2.3 Plattenkulturen ........................................................................................................ 44 2.2.4 Kultivierung in Flüssigmedien................................................................................ 44 2.2.5 Hochzelldichte-Kultivierung................................................................................... 45 2.2.6 Bestimmung der Zelldichte ..................................................................................... 45 2.2.7 Lagerung von Bakterienkulturen............................................................................. 45 VII Inhalt 2.3 Molekularbiologische Methoden.................................................................................... 46 2.3.1 Präparation von Plasmid-DNA................................................................................ 46 2.3.2 Konzentrationsbestimmung von DNA.................................................................... 46 2.3.3 Gelelektrophoretische Auftrennung von DNA (Sambrook et al., 1989) ................ 46 2.3.4 Elution von DNA aus Agarosegelen ....................................................................... 47 2.3.5 Polymerasekettenreaktion (PCR) ............................................................................ 47 2.3.5.1 Standard-PCR (nach Saiki et al., 1988) ............................................................... 47 2.3.5.2 Ortsspezifische Mutagenese................................................................................. 47 2.3.5.3 Ungerichtete Mutagenese mittels epPCR............................................................
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