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Investigation of Starch Binding Domains for Improvement of Starch Degradation

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Investigation of Starch Binding Domains for Improvement of Starch Degradation Downloaded from orbit.dtu.dk on: Dec 17, 2017 Investigation of Starch Binding Domains for Improvement of Starch degradation Christiansen, Camilla; Blennow, Anders; Viksø-Nielsen, Anders; Svensson, Birte Publication date: 2009 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Christiansen, C., Blennow, A., Viksø-Nielsen, A., & Svensson, B. (2009). Investigation of Starch Binding Domains for Improvement of Starch degradation. Kgs. Lyngby, Denmark: Technical University of Denmark (DTU). 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Investigation of Starch Binding Domains for Improvement of Starch degradation Camilla Christiansen Ph.D. Thesis February 2009 Molecular Plant Biology Department of Plant Biology and Biotechnology Faculty of Life Sciences, University of Copenhagen Investigation of Starch Binding Domains for Improvement of Starch degradation Camilla Christiansen Ph.D. Thesis February 2009 Molecular Plant Biology Department of Plant Biology and Biotechnology Faculty of Life Sciences, University of Copenhagen Preface The present Ph.D. thesis entitled “Investigation of starch binding domain for improvement of starch degradation” summarises my work carried out at Department of Plant Biology and Biotechnology, Faculty of Life Sciences at University of Copenhagen and at Department of System Biology, Enzyme and Protein Chemistry, Technical University of Denmark. The studies were financially supported by the research school for biotechnology (FOBI), Faculty of Life Sciences at University of Copenhagen and Novozymes. I would like to acknowledge my supervisors; Associate Professor Andreas Blennow and Associate Professor Lone Baunsgaard from University of Copenhagen, Professor Birte Svensson, Associate Professor Henrik Næsted and Associate Professor Maher Abou Hachem from the Technical University of Denmark, Department of Systems Biology, Science manager Anders Viksø-Nielsen and Professor Lene Lange from Novozymes. I would like to acknowledge all members – former and present – of the Plant Biochemistry Laboratory at at University of Copenhagen and Enzyme and Protein Chemistry at the Department of Systems Biology for providing a good working environment. A special thanks to Malene, Maher, Eun-Seong and Sabrina for their good spirits. Further more I would like to thank Stefan Janecek, Esben Friis, Mikkel Glaring, Lis Byrsting Møller, Morten Munch Nielsen and Martin J. Bauman for help and good collaboration during these last years and Jørgen Sauer for the original idea of fluorescence labelling the proteins. Finally my friends and family for their support during the whole period Camilla Christiansen 1 Table of contents 1 Table of contents................................................................................................................. 1 2 Summary ............................................................................................................................. 2 3 Dansk resume...................................................................................................................... 4 4 Abbreviations...................................................................................................................... 6 5 List of publications.............................................................................................................. 7 6 Introduction......................................................................................................................... 8 6.1 Starch .......................................................................................................................... 9 6.1.1 Amylose .............................................................................................................. 9 6.1.2 Amylopectin........................................................................................................ 9 6.1.3 Starch granule organisation............................................................................... 11 6.1.4 Covalently bound phosphate............................................................................. 14 6.1.5 Starch properties............................................................................................... 15 6.2 Biosynthesis of starch ............................................................................................... 15 6.3 Degradation of starch................................................................................................ 18 6.4 Glucan, water dikinases (GWDs).............................................................................. 20 6.4.1 Catalytic mechanism......................................................................................... 21 6.4.2 Homologues ...................................................................................................... 22 6.4.3 Substrate specificity.......................................................................................... 23 6.4.4 Regulation of activity........................................................................................ 24 6.5 Carbohydrate binding modules and classification .................................................... 25 6.5.1 Family CBM20................................................................................................. 25 7 Experimental work............................................................................................................ 27 7.1 Constructs, expression and purification.................................................................... 27 7.1.1 GWD3-SBD E. coli Gateway ........................................................................... 27 7.1.2 GWD3-SBD Site-directed mutagenesis............................................................ 30 7.1.3 GWD3-SBD E. coli pET28a(+)........................................................................ 30 7.1.4 GWD3-SBD P. pastoris pPICZα B .................................................................. 31 7.1.5 GWD3-SBD P. pastoris EndoH treatment ....................................................... 32 7.1.6 GA-SBD E. coli Gateway ................................................................................. 32 7.1.7 GA-SBD P. pastoris pPICZα B........................................................................ 33 7.1.8 LAF-SBD E. coli Gateway ............................................................................... 34 7.1.9 GST control expression and purification .......................................................... 34 7.1.10 GA P. pastoris pPICZα B ................................................................................. 35 7.1.11 GA without SBD P. pastoris pPICZα B ........................................................... 36 7.1.12 GA-GWD3 fusion P. pastoris pPICZα B ......................................................... 37 7.2 Homology modelling................................................................................................ 38 7.3 Differential scanning calorimetry (DSC).................................................................. 39 7.4 Surface Plasmon Resonance (SPR)........................................................................... 39 7.5 Confocal Laser Scanning Microscopy (CLSM)........................................................ 40 7.6 Transient expression of GWD3-SBD in Tobacco..................................................... 40 7.7 Starch granule binding .............................................................................................. 41 1 8 Results............................................................................................................................... 42 8.1 Production of recombinant proteins.......................................................................... 42 8.1.1 GWD3-SBD...................................................................................................... 42 8.1.2 GA-SBD and LAF-SBD ................................................................................... 46 8.1.3 GST tag............................................................................................................. 46 8.1.4 GA and GA without SBD ................................................................................. 47 8.1.5 GA-GWD3 fusion............................................................................................. 47 8.2 Sequence motifs in CBM20 ...................................................................................... 48 8.3 Homology modelling................................................................................................ 49 8.4 Stability measurements by DSC ............................................................................... 51 8.5 Affinity determined by SPR.....................................................................................
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