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Summary & Conclusions Enzymatic functionalization and degradation of natural and synthetic polymers Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Shohana Subrin Islam M. Sc. Biotechnologie aus Dhaka, Bangladesch Berichter: Univ. -Prof. Dr. Ulrich Schwaneberg Univ. -Prof. Dr. Lothar Elling Tag der mündlichen Prüfung: 23.01.2019 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar. To my mom & my sister-the two persons in the world who always stand by me Table of content Table of content Table of content _______________________________________________________________ v Publications and patents ________________________________________________________ ix Abstract _____________________________________________________________________ xi 1. General introduction _______________________________________________________ 1 1.1 Enzymatic functionalization of (bio)polymers _______________________________________ 1 1.2 Enzymatic degradation of polymers _______________________________________________ 3 1.3 Protein engineering ____________________________________________________________ 5 1.3.1 Directed evolution of enzymes _________________________________________________________ 6 1.3.2 KnowVolution – Directed Evolution 2.0 __________________________________________________ 9 1.4 Aims of the dissertation _______________________________________________________ 11 2. Engineering of an aryl sulfotransferase toward sulfation of saccharides ____________ 13 2.1 Declaration __________________________________________________________________ 13 2.2 State of the art _______________________________________________________________ 13 2.2.1 Sulfation in nature _________________________________________________________________ 13 2.2.2 Sulfation of glycosaminoglycans_______________________________________________________ 14 2.2.3 Chemical sulfation of (poly)saccharides _________________________________________________ 15 2.2.4 Classes of sulfotransferases __________________________________________________________ 16 2.2.5 Aryl sulfotransferases from Desulfitobacterium hafniense __________________________________ 19 2.2.6 Directed evolution of sulfotransferases _________________________________________________ 20 2.2.7 Objectives ________________________________________________________________________ 21 2.3 Material and methods _________________________________________________________ 22 2.3.1 Chemicals ________________________________________________________________________ 22 2.3.2 Cloning of astA and astB _____________________________________________________________ 22 2.3.3 Construction of astB SeSaM library ____________________________________________________ 23 2.3.4 Site-saturation and site-directed mutagenesis ___________________________________________ 25 2.3.5 Cultivation of libraries in 96-well microtiter plates ________________________________________ 25 2.3.6 pNPS based screening system for 96-well microtiter plates _________________________________ 26 2.3.7 Production in shaking flasks __________________________________________________________ 26 2.3.8 Purification of ASTB-WT and variants __________________________________________________ 27 2.3.9 Kinetic characterization of ASTB-WT and variants _________________________________________ 27 2.3.10 Evaluation of pH activity profile and pH stability ASTB-WT and variants ____________________ 27 2.3.11 Determination of organic solvent resistance of ASTB-WT and variants _____________________ 28 2.3.12 Determination of thermostability of ASTB-WT and variants ______________________________ 28 2.3.13 Computational analysis of ASTB homology model ______________________________________ 28 v Table of content 2.3.14 Synthesis of (mono)sulfated GlcNAc-linker-tBoc _______________________________________ 29 2.3.15 Analytical and semi-preparative HPLC _______________________________________________ 29 2.3.16 Mass spectrometry (MS) __________________________________________________________ 30 2.3.17 Nuclear magnetic resonance (NMR) _________________________________________________ 31 2.4 Results _____________________________________________________________________ 32 2.4.1 Selection of an aryl sulfotransferase ___________________________________________________ 32 2.4.2 Development of a continuous pNPS based screening system ________________________________ 33 2.4.3 Directed evolution of ASTB toward GlcNAc ______________________________________________ 35 2.4.3.1 Generation and screening of astB SeSaM library _________________________________ 36 2.4.3.2 Screening of ASTB SeSaM library ______________________________________________ 38 2.4.3.3 Kinetic characterization of ASTB-WT and variants for GlcNAc _______________________ 39 2.4.4 KnowVolution campaign of ASTB towards cellobiose ______________________________________ 40 2.4.4.1 Phases of KnowVolution _____________________________________________________ 40 2.4.4.2 Production and purification of ASTB-WT and selected variants ______________________ 44 2.4.4.3 Kinetic characterization of ASTB-WT and improved variants ________________________ 45 2.4.4.4 Determination of sulfated cellobiose monosulfate ________________________________ 46 2.4.4.5 ASTB Activity toward mono-, di-, and oligosaccharides ____________________________ 47 2.4.4.6 Sulfation of GlcNAc-linker-tBoc by ASTB-WT and improved variants __________________ 48 2.4.4.7 pH activity and pH stability ASTB-WT and improved variants ________________________ 52 2.4.4.8 Organic co-solvent activity of ASTB-WT and improved variants ______________________ 53 2.4.4.9 Thermostability of ASTB-WT and improved variants _______________________________ 55 2.5 Discussion __________________________________________________________________ 56 3. Engineering of a cutinase toward degradation of synthetic polymers _______________ 61 3.1 Declaration _________________________________________________________________ 61 3.2 State of the art ______________________________________________________________ 61 3.2.1 Plastics and their applications ________________________________________________________ 61 3.2.2 Environmental pollution by microplastic ________________________________________________ 62 3.2.2.1 Polyethylene terephthalate (PET) _____________________________________________ 64 3.2.2.2 Polyurethane (PUR) ________________________________________________________ 65 3.2.3 Biodegradation of PET and PUR _______________________________________________________ 66 3.2.4 Cutinases from Thermomonospora curvata (Tcur0390/Tcur1278) ____________________________ 67 3.2.5 Directed polymer degradation by binding domains _______________________________________ 69 3.2.6 Objectives ________________________________________________________________________ 72 3.3 Material and methods ________________________________________________________ 73 3.3.1 Chemicals ________________________________________________________________________ 73 3.3.2 Use of eGFP-anchor fusion protein for binding tests _______________________________________ 73 3.3.3 Construction of cutinase-anchor fusion protein __________________________________________ 74 3.3.4 Production of Tcur1278-WT and Tcur1278-anchors in Pichia pastoris _________________________ 74 3.3.5 Determination of cutinase activity via pNPB assay ________________________________________ 75 3.3.6 Evaluation of polyester-PUR degradation _______________________________________________ 75 vi Table of content 3.3.7 Evaluation of PET-film degradation ____________________________________________________ 75 3.3.8 Dynamic light scattering (DLS) ________________________________________________________ 76 3.3.9 Field emission scanning electron microscopy (FE-SEM) ____________________________________ 76 3.4 Results _____________________________________________________________________ 77 3.4.1 Selection of anchors for binding to polyester-PUR and PET _________________________________ 77 3.4.2 Cultivation of Tcur1278-WT and Tcur1278-anchors in Pichia pastoris _________________________ 78 3.4.3 Determination of polyester-PUR degradation ____________________________________________ 79 3.4.4 Specific activity of Tcur1278-WT and Tcur1278-TA2 _______________________________________ 80 3.4.5 Quantification of polyester-PUR degradation ____________________________________________ 81 3.4.5.1 MTP-based turbidity assay ___________________________________________________ 81 3.4.5.2 DLS of polyester-PUR particles ________________________________________________ 84 3.4.5.3 FE-SEM analysis of polyester-PUR degradation __________________________________ 85 3.4.6 Broadening the targeted degradation by anchor peptides to PET ____________________________ 86 3.5 Discussion ___________________________________________________________________ 87 4. Summary and conclusion __________________________________________________ 93 5. References ______________________________________________________________ 97 6. Appendix ______________________________________________________________ 109 6.1 List of abbreviation __________________________________________________________ 109 6.2 List of DNA and protein sequences _____________________________________________ 111 6.3 List of oligonucleotides _______________________________________________________ 118 6.4 List of computational data ____________________________________________________ 121 6.5 List of analytical data _________________________________________________________
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