Click here to load reader

Production of prebiotic exopolysaccharides by lactobacilli · PDF fileProduction of prebiotic exopolysaccharides by lactobacilli Markus Tieking Vollständiger Abdruck der von der Fakultät

  • View
    2

  • Download
    0

Embed Size (px)

Text of Production of prebiotic exopolysaccharides by lactobacilli · PDF fileProduction of prebiotic...

  • Lehrstuhl für Technische Mikrobiologie

    Production of prebiotic exopolysaccharides by lactobacilli

    Markus Tieking

    Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für

    Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung

    des akademischen Grades eines

    Doktor - Ingenieurs

    genehmigten Dissertation.

    Vorsitzender: Univ.-Prof. Dr.- Ing. E. Geiger

    Prüfer der Dissertation: 1. Univ.-Prof. Dr. rer. nat. habil. R. F. Vogel

    2. Univ.-Prof. Dr.- Ing. D. Weuster-Botz

    Die Dissertation wurde am 09.03.2005 bei der Technischen Universität eingereicht und durch

    die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt

    am 27.05.2005 angenommen.

  • Lehrstuhl für

    Technische Mikrobiologie

    Production of prebiotic exopolysaccharides by lactobacilli

    Markus Tieking

    Doctoral thesis

    Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt

    Freising 2005

  • Mein Dank gilt

    meinem Doktorvater Prof. Rudi Vogel für die Überlassung des Themas sowie die stete Diskussionsbereitschaft, Dr. Michael Gänzle für die kritische Begleitung, die ständige Diskussionsbereitschaft sowie sein fachliches Engagement, welches weit über das übliche Maß hinaus geht, Dr. Matthias Ehrmann für seine uneingeschränkte Bereitwilligkeit, sein Wissen auf dem Gebiet der Molekularbiologie weiterzugeben, seine unschätzbar wertvollen praktischen Ratschläge auf diesem Gebiet sowie für seine Geduld, meiner lieben Frau Manuela, deren Motivationskünste und emotionale Unterstützung mir wissenschaftliche und experimentelle „Durststrecken“ zu bewältigen halfen, der Ernst Böcker GmbH und dem Fleischmann Fonds für die finanzielle Unterstützung, meinen Labortisch- und Büronachbarn Clarissa Schwab, Nicoline Vermeulen und Dirk Margosch für die freundschaftliche und humorvolle Arbeitsatmosphäre trotz chronischem Platzmangels und zu heißen Labortemperaturen, Georg Maier für seine Hilfsbereitschaft, seinen Fleiß und Einsatz, all den Kollegen an der TMW für das konstruktive Miteinander und das kollegiale Verhältnis, all den Student(in)en, die ich im Rahmen von Diplom- und Semesterarbeiten betreut habe für die meist engagierte und interessierte Mitarbeit.

  • Abbreviations frequently used in this thesis:

    aa amino acid

    AFLP Amplified Fragment Length

    Polymorphism

    bp base pairs

    cfu colony forming units

    dNTP desoxynucleoside triphosphate

    dTDP desoxythymidine diphosphate

    DP degree of polymerization

    EMBL European Molecular Biology

    Laboratory

    EPS exopolysaccharide

    FOS fructooligosaccharide

    GOS glucooligosaccharide

    HMW high molecular weight

    HoPS homopolysaccharide

    HePS heteropolysaccharide

    HeOS heterooligosaccharide

    HPAEC-IPAD High Performance Anion Exchange

    Chromatography with Integrated

    Pulsed Amperometric Detection

    HPLC High Performance Liquid

    Chromatography

    LAB lactic acid bacteria

    LMW low molecular weight

    ORF open reading frame

    PAGE polyacrylamide gel electrophoresis

    PCR polymerase chain reaction

    GlcNAc N-acetyl-glucosamine

    GalNAc N-acetyl-galactosamine

    GPC gel permeation chromatography

  • RAPD Randomly Amplified Polymorphic

    DNA

    RI refractive index

    RT-PCR reverse transcrictase PCR

    SDS sodium dodecyl sulfate

    UDP undecaprenylphosphate

    WS-PS water soluble polysaccharides

  • Contents

    1 Introduction 1

    1.1 Lactic acid bacteria in cereal fermentations 1

    1.2 Exopolysaccharides from lactic acid bacteria 1

    1.3 Glucan and fructan biosynthetic enzymes 6

    1.4 Application of EPS from lactobacilli: overview 8

    1.5 Motivation and objectives 9

    2 Materials and Methods 11

    2.1 Strains, media and growth conditions 11

    2.2 EPS-screening 11

    2.2.1 Screening of cereal associated lactobacilli for EPS synthesis 11

    2.2.2 Screening of lactobacilli isolated from ducks for EPS synthesis 13

    2.2.3 Isolation of lactobacilli from pig feces and screening for EPS

    production 13

    2.2.4 Monosaccharide composition of EPS 14

    2.2.5 Preparation of doughs for the purpose of EPS screening 14

    2.3 High Performance Liquid Chromatography (HPLC)-analytics 15

    2.4 Molecular techniques 16

    2.4.1 General molecular techniques 16

    2.4.2 Construction of primers targeting fructosyl- and glucosyltransferases 19

    2.4.3 Isolation of mRNA and RT-RCR 19

    2.4.4 Amplified Fragment Length Polymorphism (AFLP) 20

    2.4.5 Plasmids 21

    2.4.6 Nucleotide sequence analysis of the levansucrase gene sequence from L.

    sanfranciscensis TMW 1.392 21

    2.4.7 Nucleotide sequence analysis of the glucansucrase gene sequence from L.

    animalis TMW 1.971 21

    2.4.8 Cloning, heterologous expression and purification of the levansucrase 22

    2.4.9 Insertional inactivation of the levansucrase gene by single crossover

    integration 23

  • 2.4.10 Isolation of RNA from dough fermented with L. sanfranciscensis TMW

    1.392 and TMW 1.392∆lev, RT-RCR and amplification of a levansucrase and

    phosphotransacetylase internal fragment from cDNA 25

    2.5 Characterization of the sucrose metabolism of L. sanfranciscensis

    TMW 1.392 and TMW 1.392∆lev in medium and doughs 26

    2.6 Characterization of the levansucrase from L. sanfranciscensis 27

    2.7 Partial purification and characterization of oligosaccharides from enzymatic

    reactions and sourdoughs 28

    2.8 Chromatographic separation of oligosaccharides 29

    3 Results 30

    3.1 In situ production of exopolysaccharides during sourdough fermentation

    by cereal and intestinal isolates of lactic acid bacteria 30

    3.1.1 Screening of sourdough lactobacilli for EPS production 30

    3.1.2 PCR-screening for a levansucrase gene and sequence of the

    amplification product 33

    3.1.3 Comparison of the sequence of levV-levR PCR-products with other

    bacterial levansucrases 34

    3.1.4 Expression of the levansucrase gene in L. sanfranciscensis 36

    3.1.5 In situ EPS-formation in sourdough 37

    3.2 Molecular and functional characterization of a levansucrase from

    Lactobacillus sanfranciscensis 40

    3.2.1 Nucleotide sequence analysis of the L. sanfranciscensis TMW1.392

    levansucrase gene 40

    3.2.2 Analysis of the amino acid (aa)sequence and alignments with bacterial

    levansucrases 41

    3.2.3 Insertional inactivation of the levansucrase gene and sucrose

    metabolism by the levansucrase deficient strain 43

    3.2.4 Dough fermentations with the wild type strain and the levansucrase

    deficient strain. 46

    3.2.5 Heterologous expression and biochemical characterization of the

    recombinant enzymes. 50

  • 3.3 Evidence for formation of heterooligosaccharides (HeOS) by Lactobacillus

    sanfranciscensis during growth in wheat sourdough. 56

    3.3.1 Enzymatic synthesis of fructooligosaccharides (FOS) and HeOS 56

    3.3.2 FOS and HeOS from sucrose or raffinose as fructosyl-donor and

    –acceptor 57

    3.3.3 HeOS from maltose and maltotriose as fructosyl-acceptors 58

    3.3.4 HeOS from xylose or arabinose as fructosyl-acceptors 60

    3.3.5 Oligosaccharides formed during sourdough fermentations with

    L. sanfranciscensis 61

    3.4 Extracellular homopolysaccharides and oligosaccharides from intestinal

    lactobacilli 64

    3.4.1 Screening of lactobacilli isolated from ducks for HoPS synthesis 64

    3.4.2 Production of oligosaccharides by HoPS-producing strains 64

    3.4.3 Molecular screening for genes encoding glucosyl- and

    fructosyltransferases 66

    3.4.4 Identification of lactobacilli from the pig feces and their ability

    for HoPS formation 69

    3.5 Isolation and nucleotide sequence analysis of the L. animalis strain 1.971

    glucansucrase gene 72

    3.5.1 Nucleotide sequence analysis 72

    3.5.2 Amino acid sequence analysis 73

    4 Discussion 75

    4.1 Screening of cereal lactobacilli for EPS synthesis and genes coding for EPS

    synthesizing genes 75

    4.2 Isolation and characterization of a levansucrase from L. sanfranciscensis

    TMW 1.392 79

    4.3 Heterooligosaccharides in sourdough fermentations 83

    4.4 Screening of lactobacilli isolated from the intestinum of ducks and pigs 86

    4.5 Molecular characterization of a glucansucrase from L. animalis 88

    5 Summary and concluding remarks 89

    6 Zusammenfassung und Ausblick 93

    7 References 97

    8 List of publications 111

  • 1

    1 Introduction

    1.1 Lactic acid bacteria in cereal fermentations

    Following a long tradition, sourdough is used as leavening agent in bread production.

    Sourdough fermentations as well as baking agents based on sourdoughs retained their

    importance in contemporary baking technology because of the improve

Search related