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Dissertation.Pdf Gentechnisches Enzymdesign zur Verbesserung der Eigenschaften von Isomaltulose-Synthase aus Protaminobacter rubrum Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Susann Baumert aus Osterburg 1. Referent: Professor. Dr. Klaus-Dieter Vorlop 2. Referent: apl. Professor Siegmund Lang eingereicht am: 19.12.2011 mündliche Prüfung (Disputation) am: 20.04.2012 Druckjahr 2012 Inhaltsverzeichnis Inhaltsverzeichnis Abbildungsverzeichnis ........................................................................................................... iii Tabellenverzeichnis ............................................................................................................... iv Symbol- und Abkürzungsverzeichnis ...................................................................................... v 1 Einleitung und Zielsetzung ...................................................................................... 1 2 Theoretische Grundlagen ........................................................................................ 3 2.1 Palatinose und ihre Eigenschaften .......................................................................... 3 2.1.1 Verwendung von Palatinose in der Lebensmittelindustrie ................................. 4 2.1.2 Verwendung der Palatinose als Ausgangsstoff für die chemische Industrie ..... 5 2.1.3 Industrieller Herstellungsprozess...................................................................... 5 2.2 Das Enzym Isomaltulose-Synthase ......................................................................... 7 2.2.1 Klassifizierung und Nomenklatur der Isomaltulose-Synthase ........................... 7 2.2.2 Die Reaktion der Isomaltulose-Synthase .......................................................... 7 2.2.3 Isomaltulose-Synthase bildende Mikroorganismen ........................................... 8 2.2.4 Produktselektivität der Isomaltulose-Synthase ................................................. 9 2.2.5 Strukturelle Kenntnisse der Isomaltulose-Synthase .........................................10 2.3 Gentechnisches Enzymdesign ...............................................................................13 2.3.1 Gerichtete Evolution ........................................................................................13 2.3.2 Rationales Proteindesign ................................................................................14 2.3.3 Semirationales Proteindesign ..........................................................................15 2.3.4 Screening- und Selektionsmethoden ...............................................................16 2.3.5 Expressionssystem auf Basis von Escherichia coli ..........................................18 2.4 Thermostabilität von Enzymen ...............................................................................19 3 Material und Methoden ..........................................................................................23 3.1 Verwendete E. coli -Stämme ...................................................................................23 3.1.1 Wirtsstamm JM105..........................................................................................23 3.1.2 pMSpal127 ......................................................................................................23 3.2 Kultivierungsbedingungen ......................................................................................24 3.2.1 Verwendete Medien ........................................................................................24 3.2.2 Kultivierung in 5-mL-Kulturröhrchen ................................................................25 3.2.3 Kultivierung im Schüttelkolben ........................................................................26 3.3 Analytik ..................................................................................................................27 3.3.1 Bestimmung der optischen Dichte (OD 600 ) .......................................................27 3.3.2 High performance liquid chromatography (HPLC) ...........................................27 3.3.3 Bestimmung der Plasmid-DNA-Menge ............................................................29 3.3.4 Bestimmung der löslichen Proteinkonzentration ..............................................30 3.3.5 Semiquantitative Erfassung der Proteine durch SDS-PAGE ............................31 3.4 Zellaufschluss ........................................................................................................32 3.5 Standard-Enzymaktivität ........................................................................................33 3.5.1 Berechnung der Enzymaktivität der Isomaltulose-Synthase ............................34 3.6 Analyse des Produktspektrums der Isomaltulose-Synthase ...................................34 3.7 Analyse der Thermostabilität ..................................................................................35 3.8 In vitro DNA-Rekombination (Klonierung) zur Herstellung der Deletionsmutanten – SFI und –FIE ..........................................................................................................35 3.8.1 Site-directed Mutagenese von Plasmid-DNA durch die Polymerasekettenreaktion (PCR) ....................................................................36 3.8.2 Restriktionsverdau mit DpnI ............................................................................37 3.8.3 Ligation der linearen PCR-Produkte ................................................................38 3.8.4 Herstellung chemo-kompetenter JM105-Zellen ...............................................38 3.8.5 Transformation durch Hitzeschock ..................................................................38 3.8.6 Identifizierung der Mutanten ............................................................................39 4 Ergebnisse und Diskussion ....................................................................................40 i Inhaltsverzeichnis 4.1 Funktionelle Charakterisierung der physiologischen Eigenschaften der PalI-Enzyme aus den rekombinanten Mutanten ..........................................................................40 4.1.1 Screening der besten Kandidaten ...................................................................41 4.1.2 Wachstumsverhalten .......................................................................................42 4.1.3 Aufnahme des Produktspektrums ....................................................................43 4.1.4 Temperatureinfluss auf die PalI-Selektivität .....................................................45 4.2 Deletionsmutanten .................................................................................................48 4.2.1 Auswahl der Klonierungsstrategie ...................................................................48 4.2.2 Selektion der Deletionsmutanten .....................................................................49 4.2.3 Auswirkung der Deletionen auf die Eigenschaften des rekombianten PalI- Enzyms ...........................................................................................................51 4.2.4 Zusammenfassung: Effekte der Deletionen auf die Eigenschaften des rekombinanten PalI-Enzyms ............................................................................61 4.3 Substitutionsmutanten............................................................................................63 4.3.1 Deskription der Substitutionsmutanten ............................................................63 4.3.2 Untersuchung zur Voraussage von Effekten definierter Mutationen in homologen Enzymen .......................................................................................67 4.3.3 Auswirkung der Substitutionen auf die Eigenschaften des rekombianten PalI- Enzyms ...........................................................................................................71 4.3.4 Zusammenfassung: Effekte der AS-Substitutionen auf die Eigenschaften des PalI-Enzyms ....................................................................................................82 5 Zusammenfassung ................................................................................................84 6 Schlussfolgerung und Ausblick ..............................................................................85 7 Literaturverzeichnis ................................................................................................87 8 Anhang ..................................................................................................................98 Danksagung ....................................................................................................................... 103 ii Abbildungsverzeichnis Abbildungsverzeichnis Abbildung 2-1 (a) Chemische Struktur der Saccharose; (b) Chemische Struktur der Palatinose ................................................................................................. 3 Abbildung 2-2 Enzymatische Reaktion der Isomaltulose-Synthase nach Zhang et al., 2003 ......................................................................................................... 8 Abbildung 2-3 Übereinander gelegte 3D-Struktur der Isomaltulose-Synthase aus (Lipski et al., 2010) ...................................................................................11 Abbildung 2-4 Abhängigkeit der Enzymaktivität von der Temperatur...............................20
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