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Charakterisierung Lebensmitteltechnologisch Relevanter Ferulasäureesterasen Aus Basidiomycota

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Charakterisierung Lebensmitteltechnologisch Relevanter Ferulasäureesterasen Aus Basidiomycota Charakterisierung lebensmitteltechnologisch relevanter Ferulasäureesterasen aus Basidiomycota Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte Dissertation von M.Sc. Annabel Nieter geboren am 06.01.1988 in Halberstadt 2016 Inhaltsverzeichnis Referent: Prof. Dr. rer. nat. Dr.-Ing. Ralf Günter Berger Korreferent: Prof. Dr. rer. nat. Thomas Scheper Tag der Promotion: 02.03.2016 Danksagung Mein besonderer Dank gilt meinem Doktorvater Herrn Prof. Dr. Dr. Ralf G. Berger für die wissenschaftliche Betreuung dieser Arbeit, die stete Diskussionsbereitschaft, das entgegengebrachte Vertrauen und der gewährten Freiheit bei der Ausgestaltung der experimentellen Arbeit. Darüber hinaus möchte ich mich für die Bereitstellung der hervorragenden Arbeitsbedingungen am Institut sowie der Unterstützung bei der Veröffentlichung meiner Ergebnisse in Fachjournalen bedanken. Herzlichst danken möchte ich zudem Frau Dr. Diana Linke für ihre herausragende Betreuung und ihr offenes Ohr für Anliegen aller Art. Ihre Anregungen, wertvollen Tipps und geduldigen Erklärungen haben entscheidend zum erfolgreichen Gelingen dieser Arbeit beigetragen. Herrn Prof. Dr. Thomas Scheper (Institut für technische Chemie, Leibniz Universität Hannover) danke ich für die freundliche Übernahme des Korreferats und Herrn Prof. Dr. Thomas Debener (Institut für Pflanzengenetik, Leibniz Universität Hannover) für die Bereitschaft, den Vorsitz meiner Disputation zu übernehmen. Dem BMBF Cluster BIOKATALYSE2021 danke ich für die finanzielle Unterstützung des Projektes „Entwicklung und Optimierung eines mikrobiellen Expressionssystems zur Produktion von Feruloyl- Esterasen“ (P37), in dessen Rahmen diese Arbeit angefertigt wurde. Des Weiteren gilt mein ausdrücklicher Dank Herrn Prof. Dr. Mirko Bunzel und seinen Mitarbeitern (Institut für Angewandte Biowissenschaften, Karlsruher Institut für Technologie (KIT)) für die Bereitstellung der feruloylierten Saccharide. Herrn Dr. Manfred Nimtz (Helmholtz-Zentrum für Infektionsforschung GmbH, Braunschweig) und Herrn PD Dr. Ulrich Krings danke ich für die Sequenzierung von Peptiden mittels ESI-MS/MS. Weiterhin möchte ich mich bei Herrn Dr. Lutz Popper und seinen Kollegen (SternEnzym GmbH & Co KG, Ahrensburg) für die Durchführung der rheologischen Messungen und Backversuche bedanken. Außerdem gilt mein ausdrücklicher Dank allen Kolleginnen und Kollegen am Instituts für Lebensmittelchemie für das angenehme und freundschaftliche Arbeitsklima und die hilfsbereite Zusammenarbeit. Die gemeinsam verbrachte Zeit hat mir viel Freude bereitet. Nicht zuletzt möchte ich besonders meinen Eltern danken, die mich in meinem Leben immer unterstützt und gefördert haben, meinen Wünschen und Plänen gegenüber offen waren und mir jederzeit mit Rat und Tat zur Seite standen. Vorbemerkung Diese Dissertationsschrift basiert auf Arbeiten, die in folgenden peer reviewed Publikationen veröffentlicht wurden: . Nieter, A.; Haase-Aschoff, P.; Linke, D.; Nimtz, M.; Berger, R. G.; (2014). „A halotolerant type A feruloyl esterase from Pleurotus eryngii”. Fungal Biology, 118, 348-357. Nieter, A.; Haase-Aschoff, P.; Kelle, S.; Linke, D.; Krings, U.; Popper, L.; Berger, R. G.; (2015). „A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis”. Applied and Environmental Microbiology, 81(5), 1679-1688. Nieter, A.; Kelle, S.; Takenberg, M.; Linke, D.; Bunzel, M.; Popper, L.; Berger, R. G.; (2016). „Heterologous production and characterization of a chlorogenic acid esterase from Ustilago maydis with a potential use in baking”. Food Chemistry, 209, 1-9. Für einen Teil dieser Arbeit wurde ein Patent eingereicht: . Nieter, A.; Linke, D.; Berger, R. G.; Thiesing, D.; Popper, L.; (2014). “Chlorogenic acid esterases with a distinct feruloyl esterase side activity”. Appl. No. EP14182006.8. Nachfolgend gelistete Veröffentlichungen und Poster wurden im Rahmen dieser Doktorarbeit erarbeitet, sind jedoch nicht Gegenstand dieser Dissertationsschrift. Veröffentlichung: . Kelle, S.; Nieter, A.; Krings, U.; Zelena, K.; Linke, D.; Berger, R. G.; (2015). „Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl-saccharide esters”. Biotechnology and Applied Biochemistry, DOI 10.1002/bab.1430. Posterpräsentation: . Nieter, A.; Linke, D.; Berger, R. G.; “A unique esterase from Ustilago maydis catalyzes the hydrolysis of chlorogenic acid and feruloylated saccharides”. 7th International Congress on Biocatalysis, Hamburg, 31. August – 04. September 2014. Inhaltsverzeichnis I Inhaltsverzeichnis Inhaltsverzeichnis ........................................................................................................................................... I Abkürzungsverzeichnis ............................................................................................................................... IV Zusammenfassung ..................................................................................................................................... VII Abstract ..................................................................................................................................................... VIII 1. Einleitung ............................................................................................................................................... 1 1.1 Pflanzliche Zellwände als Ressource erneuerbarer Energien ......................................................... 1 1.1.1 Vorkommen von Phenolsäuren in pflanzlichen Zellwänden .................................................. 2 1.1.2 Bedeutung pflanzlicher Hydroxyzimtsäuren für die Industrie ............................................... 6 1.1.3 Industrielle Nebenströme als Quelle für Hydroxyzimtsäuren ................................................ 9 1.2 Ferulasäureesterasen (FAE) .......................................................................................................... 11 1.2.1 FAE aus Basidiomycota ....................................................................................................... 12 1.2.2 Klassifizierung von FAE ...................................................................................................... 14 1.2.3 Struktur und katalytischer Mechanismus von FAE .............................................................. 17 1.2.4 Industrielle Applikationen von FAE .................................................................................... 18 1.2.4.1 Papier- und Zellstoffindustrie ........................................................................................... 19 1.2.4.2 Produktion von Bioethanol ............................................................................................... 20 1.2.4.3 Hydrolyse und Synthese von bioaktiven phenolischen Verbindungen ............................ 20 1.2.4.4 Landwirtschaft .................................................................................................................. 21 1.2.4.5 Backwarenindustrie .......................................................................................................... 22 1.3 Zielsetzung der Arbeit .................................................................................................................. 23 2. Vorwort zur Publikation „ A halotolerant type A feruloyl esterase from Pleurotus eryngii”.......24 3. A halotolerant type A feruloyl esterase from Pleurotus eryngii ...................................................... 25 3.1 Introduction .................................................................................................................................. 26 3.2 Material and methods ................................................................................................................... 27 3.2.1 Chemicals and substrates ..................................................................................................... 27 3.2.2 Cultivation of Pleurotus eryngii ........................................................................................... 27 3.2.3 Purification of PeFaeA ......................................................................................................... 28 3.2.4 SDS-PAGE ........................................................................................................................... 28 3.2.5 Peptide mass fingerprint ....................................................................................................... 29 3.2.6 cDNA synthesis .................................................................................................................... 29 3.2.7 Sequence fishing .................................................................................................................. 29 3.2.8 Activity assays...................................................................................................................... 30 3.2.9 HPLC.................................................................................................................................... 30 3.2.10 Preparative isoelectric focussing (IEF) ................................................................................ 31 3.2.11 Effects of pH, temperature, solvents, and inhibitors on enzyme activity ............................. 31 3.2.12 Substrate specificity and kinetic parameters ........................................................................ 32 II Inhaltsverzeichnis Inhaltsverzeichnis 3.2.13 Release
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