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Novel Fructans from Acetic Acid Bacteria

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Novel Fructans from Acetic Acid Bacteria TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Technische Mikrobiologie Novel fructans from acetic acid bacteria Frank Jakob Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. S. Scherer Prüfer der Dissertation: 1. Univ.-Prof. Dr. R. F. Vogel 2. Univ.-Prof. Dr. W. Liebl 3. apl. Prof. Dr. P. Köhler Die Dissertation wurde am 23.01.2014 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 15.04.2014 angenommen. VORWORT Die vorliegende Arbeit wurde durch Fördermittel des Bundesministeriums für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV) über die Bundesanstalt für Landwirtschaft und Ernährung (BLE) unterstützt (Projekt 28-1-63.001-07). Mein besonderer Dank gilt meinem Doktorvater Prof. Dr. Rudi F. Vogel für die Möglichkeit, diese Dissertation an seinem Institut durchzuführen. Zudem möchte ich mich für seine konstruktiven Anregungen zu dieser Arbeit, sein entgegengebrachtes Vertrauen, seinen ständigen Einsatz für meine Weiterbeschäftigung an seinem Institut und für seine Unterstützung, mich wissenschaftlich weiter entwickeln zu können, bedanken. Mein außerordentlicher Dank gilt ihm außerdem für sein entgegengebrachtes Verständnis in schwierigen Phasen. Bei Dr. Daniel Meißner und Dr. Susanne Kaditzky möchte ich mich für die hilfreiche und angenehme Betreuung und bei Maria Hermann für die gute Zusammenarbeit im Projekt bedanken. Mein besonderer Dank gilt zudem Stefan Steger für die Durchführung von Backversuchen. Bei Dr. Andre Pfaff und Dr. Ramon Novoa-Carballal möchte ich mich für die entspannte Kooperation, die Durchführung von NMR-Messungen und die Bereitstellung von aufgenommenen Spektren bedanken. Bei Prof. Dr. Axel H. E. Müller bedanke ich mich für die Möglichkeit, NMR Messungen an seinem Institut durchzuführen. Bei Heinrich Rübsam bedanke ich mich für die Durchführung von AF4- Messungen und die Bereitstellung von Spektren und Rohdaten. Prof. Dr. Thomas Becker danke ich für die Möglichkeit, AF4-Messungen an seinem Institut durchzuführen. Des Weiteren möchte ich mich bei Prof. Dr. Wolfgang Liebl für die Bereitstellung von Essigsäurebakterien-Stämmen bedanken. Prof. Dr. Matthias Ehrmann und Dr. Patrick Preissler möchte ich für die Ratschläge und Diskussionen rund um das molekular- und mikrobiologische Arbeiten danken. Bei Dr. Jürgen Behr bedanke ich mich für die Unterstützung bei Peptidsequenzierungen. Dr. Jasmin Stadie danke ich für Unterstützung bei HPLC Messungen und Dr. Anna Gulitz für die Isolierung von Gluconobacter species TMW 2.1191. Zudem möchte ich mich bei allen (ehemaligen) Mitarbeitern des Lehrstuhls für Technische Mikrobiologie für ihre Hilfsbereitschaft, die nette Atmosphäre und die schöne, lehrreiche Zeit bedanken. Zuletzt möchte ich mich insbesondere bei meiner Familie bedanken, die mich ständig unterstützt hat, auf die ich mich immer verlassen konnte und die mir deswegen das Leben erheblich erleichtert hat. Allen Freunden aus Freising und Franken sei zudem für die lässige Zeit außerhalb der Uni gedankt. ABBREVIATIONS A. Acetobacter aa amino acid AAB acetic acid bacteria AF4 asymmetric flow field-flow fractionation Amp ampicillin APS ammonium persulfate Asn asparagine Asp aspartic acid bp base pair C carbon cfu colony forming units cm centimeter COSY 1H-1H two-dimensional NMR homonuclear chemical shift correlation Cys cysteine Da Dalton dg degenerate DNA deoxyribonucleic acid D2O deuterium oxide DP degree of polymerization DSM Deutsche Sammlung von Mikroorganismen DTT dithiothreitol E. Escherichia EDTA ethylene diamine tetraacetic acid EPS exopolysaccharide Fig. figure FOS fructooligosaccharide FTF fructosyltransferase ftf fructosyltransferase gene g (centrifugation) relative centrifugal force g (weight) gram G. Gluconobacter Ga. Gluconacetobacter Gln glutamine Glu glutamic acid GTF glycosyltransferase H hydrogen h hour HePS heteropolysaccharide HMQC 13C-1H two-dimensional NMR; heteronuclear multiple quantum coherence HoPS homopolysaccharide HPLC high-performance liquid chromatography HPMC hydroxypropylmethylcellulose Hz Hertz IS insertion sequence K Kelvin k constant L liter L. Leuconostoc LAB lactic acid bacteria LB lysogeny broth LC-MS/MS liquid chromatography coupled to tandem mass spectrometry Leu leucine M molar m meter MALS multi-angle light scattering Mi molar mass mi modified inverse min minute mMRS modified deMan, Rogosa, Sharp medium MN number average molar mass msf modified site-finding msp modified single primer MW weight average molar mass MWCO molecular weight cut off MWi molecular weights N Newton N. Neoasaia Na natrium/sodium NaG natrium/sodium-gluconate medium NBRC biological resource center NCBI national center for biotechnology information ni number of molecules NMR nuclear magnetic resonance nt nucleotide OD optical density o/n overnight ORF open reading frame p probability PAGE polyacrylamide gel electrophoresis PAPE phage associated promoter element PBS phosphate buffered saline PCR polymerase chain reaction PDB protein data bank PDI polydispersity index ppm parts per million RGi radiuses of gyration RI refractive index rms root mean square rRNA ribosomal ribonucleic acid rpm revolutions per minute RT retention time s second SAP shrimp alkaline phosphatase SDS sodium dodecyl sulfate si standard inverse sp. species TCA tricarboxylic acid TEMED tetramethylethylendiamine Tm melting temperature TMW Technische Mikrobiologie Weihenstephan U unit UDP uridin diphosphate V volt vG hydrodynamic coefficient v/v volume/volume vs. versus w/v weight/volume w/w weight/weight Z. Zymomonas CONTENTS 1. INTRODUCTION ..............................................................................................................................................1 1.1 Phylogeny and ecology of acetic acid bacteria ............................................................................................. 1 1.2 Physiology of AAB ...................................................................................................................................... 1 1.3 Biotechnological applications of AAB and their use in foods ...................................................................... 3 1.4 Exopolysaccharide production by AAB ....................................................................................................... 3 1.5 Occurrence of fructans ................................................................................................................................. 4 1.6 Bacterial fructan biosynthesis ...................................................................................................................... 5 1.7 Applications of fructans ............................................................................................................................... 6 1.8 Fructan producing AAB ............................................................................................................................... 6 1.9 Aims of this work ......................................................................................................................................... 7 2. MATERIAL AND METHODS .........................................................................................................................8 2.1 Strains, media and growth conditions .......................................................................................................... 8 2.1.1 Acetic acid bacteria .............................................................................................................................. 8 2.1.2 Lactic acid bacteria ............................................................................................................................... 8 2.1.3 Escherichia strains ................................................................................................................................ 8 2.2 EPS recovery ................................................................................................................................................ 9 2.2.1 Conventional EPS production............................................................................................................... 9 2.2.2 Enzymatic fructan production............................................................................................................... 9 2.2.3 Enzymatic glucan production ............................................................................................................... 9 2.2.4 Heteropolysaccharide production ....................................................................................................... 10 2.3 Baking experiments .................................................................................................................................... 10 2.3.1 Baking of wheat breads ...................................................................................................................... 10 2.3.2 Volume measurements ....................................................................................................................... 10 2.3.3 Texture profile analysis (TPA) ........................................................................................................... 11 2.4 Structural analyses of EPSs .......................................................................................................................
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