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Functional Characterization of Carbohydrate-Active Enzymes from Marine Bacteria

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Functional Characterization of Carbohydrate-Active Enzymes from Marine Bacteria Functional characterization of carbohydrate-active enzymes from marine bacteria I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Greifswald vorgelegt von Marcus Bäumgen Greifswald, 28.02.2020 Dekan: Prof. Dr. Werner Weitschies 1. Gutachter: Prof. Dr. Uwe T. Bornscheuer 2. Gutachter: Prof. Dr. Harry Brumer Tag der Promotion: 24.06.2020 II III Wissenschaft ist das Werkzeug, welches es uns ermöglicht, das große Puzzel der Natur und des Lebens zu lösen. IV Auch wenn wir den Weg des Wissens und der Weisheit niemals bis zum Ende beschreiten können, so ist doch jeder Schritt, den wir tun, ein Schritt in eine bessere Welt. V Content Abbreviations ..................................................................................................................... IX 1. Introduction ..................................................................................................................... 1 1.1 The marine carbon cycle .............................................................................................. 1 1.1.1 Algal blooms .......................................................................................................... 1 1.1.2 The marine carbohydrates ulvan and xylan ........................................................... 2 1.1.3 Marine polysaccharide utilization ........................................................................... 4 1.2 Carbohydrate-active enzymes ...................................................................................... 5 1.2.1 Glycoside hydrolases ............................................................................................ 6 1.2.2 Glycosyltransferases ............................................................................................. 9 1.2.3 Polysaccharide lyases ........................................................................................... 9 1.2.4 Polysaccharide sulfatases ....................................................................................11 1.2.5 Carbohydrate esterases .......................................................................................12 1.2.6 Auxilliary activities ................................................................................................13 1.2.7 Multimodular CAZymes ........................................................................................14 1.2.8 Ulvan- and xylan-active enzymes .........................................................................14 1.3 Applications of marine polysaccharide utilization systems ...........................................16 1.4 Analytics of polysaccharide degradation .....................................................................18 2. Scope of this work ..........................................................................................................20 3. Results ............................................................................................................................22 3.1 Extraction and processing of ulvan from green algae ..................................................22 3.2 Characterization of ulvan-degrading enzymes from F. agariphila ................................23 3.2.1 CAZyme activities on ulvan from different sources ...............................................23 3.2.2 Ulvan-active polysaccharide sulfatases ................................................................26 3.2.3 Complete elucidation of an ulvan degradation pathway ........................................31 3.2.4 The alternative ulvan degradation pathway...........................................................34 3.2.5 A novel class of ulvan-active dehydratases ..........................................................36 3.3 Characterization of xylan-degrading enzymes .............................................................44 3.3.1 Xylan-degrading enzymes from Muricauda sp. .....................................................44 3.3.2 Xylan-degrading enzymes from Flavimarina sp. ...................................................45 VI 4. Discussion ......................................................................................................................63 4.1 Extraction and properties of ulvan from different sources ............................................63 4.2 Characterization of ulvan-degrading enzymes from F. agariphila ................................63 4.2.1 Investigation of CAZymes active on ulvan from different sources .........................63 4.2.2 Ulvan-active polysaccharide sulfatases ................................................................64 4.2.3 Complete elucidation of an ulvan degradation pathway ........................................66 4.2.4 The alternative ulvan degradation pathway...........................................................68 4.2.5 A novel class of ulvan-active dehydratases ..........................................................69 4.3 Characterization of xylan-degrading enzymes .............................................................71 4.3.1 Xylan-degrading enzymes from Muricauda sp. .....................................................71 4.3.2 Xylan-degrading enzymes from Flavimarina sp. ...................................................72 4.4 Outlook .......................................................................................................................74 5. Conclusion ......................................................................................................................76 6. Materials and Methods ...................................................................................................78 6.1 Materials .....................................................................................................................78 6.1.1 Programms ...........................................................................................................78 6.1.2 Devices ................................................................................................................78 6.1.3 Chemicals and consumables ................................................................................81 6.1.4 Enzymes ..............................................................................................................81 6.1.5 Plasmids ...............................................................................................................81 6.1.6 Strains ..................................................................................................................81 6.1.7 Oligonucleotides ...................................................................................................82 6.1.8 Media and additives ..............................................................................................83 6.1.9 Buffers and solutions ............................................................................................84 6.2 Methods ......................................................................................................................86 6.2.1 Microbiological methods .......................................................................................86 6.2.2 Molecularbiological methods ................................................................................87 6.2.3 Biochemical methods ...........................................................................................90 6.2.4 Analytical methods ...............................................................................................92 7. Literature .....................................................................................................................93 VII 8. Appendix ....................................................................................................................... 110 Affirmation ........................................................................................................................ 116 List of Publications .......................................................................................................... 117 Acknowledgements .......................................................................................................... 118 VIII Abbreviations % per cent °C degree celcius Δ 4,5-unsaturated uronic acid residue or difference µg microgramm µL microliter µM micromolar A2XX 2-O-α-L-arabinofuranosyl-xylotriose A3X 3-O-α-L-arabinofuranosyl-xylobiose AA auxiliary activities a. dest. distilled water Amp ampicillin APS ammonium persulfate Ara L-arabinose ATP adenosine triphosphate Å ångström B. licheniformis Bacillus licheniformis bp base pairs BCA bicinchoninic acid BSA bovine serum albumin c concentration CAZyme carbohydrate-active enzyme CBM carbohydrate-binding module CE carbohydrate esterase C-PAGE carbohydrate polyacrylamid gel electrophoresis Da dalton DMSO dimethyl sulfoxide DNA desoxyribonucleic acid DOM dissolved organic matter E. coli Escherichia coli EDTA ethylenediaminetetraacetic acid e.g. for example EPS extracelluar polymeric substances IX et al. et alia F. agariphila Formosa agariphila FACE fluorophore-assisted carbohydrate electrophoresis FAD flavin adenine dinucleotide FGE formylglycine-generating enzyme fGly formylglycine fw forward g gramm or gravitational acceleration Gal D-galactose GC/MS gas chromatography coupled with mass spectrometry GH glycoside hydrolase GlcA D-glucuronic acid GT glycosyltransferase h hour HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
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