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Bacterial Branching Enzymes As Agents for Modifying Glucan Structure in Industrial Processing

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Bacterial Branching Enzymes As Agents for Modifying Glucan Structure in Industrial Processing Bacterial Branching Enzymes as Agents for Modifying Glucan Structure in Industrial Processing by Lily Nasanovsky A Thesis presented to The University of Guelph In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular and Cellular biology Guelph, Ontario, Canada © Lily Nasanovsky, September, 2017 ABSTRACT Bacterial Branching Enzymes as Agents for Modifying Glucan Structure in Industrial Processing Lily Nasanovsky Advisor: University of Guelph, 2017 Dr. Ian J. Tetlow Starch is used as a cheap, renewable, chemically-reactive matrix in many industrial processes. During processing, access to chemically-reactive groups on starch is essential and largely depends on their exposure, which is in part, a function of the branching frequency within starch. The amylose component of starch increases retrogradation (gelling property) of the cooked polymer at lower temperatures, forcing industries to use high temperatures throughout processing. The ability to manipulate glucan branching in starch and other polyglucans and remove amylose, offers many industrial end users (e.g. paints/inks, coatings, adhesives sectors) with superior performance bio-products. Branching enzymes (BEs) introduce α-1,6 branch points in starch and thus increase the number of reactive non-reducing chemical groups making post-harvest starch more chemically-reactive, facilitating its solubility and reducing retrogradation. Starch-derived polyglucans tend to gel rapidly, particularly in the presence of linear amylose chains, which hinders industrial processing and reduces polyglucan usability. BEs are promising industrial tools for increasing branch frequency and producing starches with improved physicochemical properties, and for reducing glucan chain length (by removing amylose) and alleviating retrogradation, and thus improving the solubility properties of post- harvest starch. Presented here is a detailed biochemical and functional characterization of recombinant glycogen branching enzymes from Thermus thermophilus and Deinococcus radiodurans (DrGBE). Additionally, the presented work shows a proof of concept and demonstrates a novel application of DrGBE to modify a commercial starch-based polyglucan to reduce gelling, improve stability and solubility, and produce a visco-stable product. ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Ian Tetlow, for his continuous support and guidance throughout my PhD work. I could not thank you enough for listening to me and guiding me through the hard times. I would also like to express my gratitude to all the past and present members of the Emes/Tetlow lab for their pertinent advice and moral support. A special thank you to Dr. Amina Makhmoudova for the valuable discussions, suggestions and constant help. I would like to extend a special thank you to all my friends who have been there for me throughout this life-changing journey. Especially, my besties Danve Castroverde and Emily Padhi, who have lent me an ear during difficult times, always offered sincere support and valuable suggestions. I love and appreciate you guys, and I’m looking forward to many more shared adventures. A very warm and special thank you to my love, Martin, who has always supported me and helped me all throughout. Words cannot express the feeling of my love, devotion, thanks and gratitude to you. You mean the world to me. Finally, I would like to thank my parents who have been an incredible source of inspiration and a solid support for me throughout my life. Thank you for loving, encouraging, and pushing me to make my dreams come true. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................... iv LIST OF FIGURES ...................................................................................................... viii LIST OF TABLES ......................................................................................................... xi LIST OF ABBREVIATIONS ....................................................................................... xii Chapter 1 - General Introduction ..................................................................................... 1 1.1 Introduction ............................................................................................................... 1 1.2 Starch and Glycogen Structure .................................................................................. 4 1.2.1 Starch and Glycogen Synthesis ............................................................................ 10 1.2.2 Mode of Action and Properties of Branching Enzymes ....................................... 16 1.3 Structure and Function Relationships of Branching Enzymes and Their Protein Domains ......................................................................................................................... 22 1.3.1 The Central Catalytic Domain .............................................................................. 24 1.3.2 The Amino (NH2-) Terminal Domain .................................................................. 31 1.3.3 The Carboxy-Terminal Domain ........................................................................... 34 1.4 Post-Harvest Starch Modifications and Applications ............................................. 37 1.4.1 Chemical Modification of Starch ......................................................................... 38 1.4.2 Physical Modifications of Starch ......................................................................... 44 1.4.3 Enzymatic Modifications of Starch ...................................................................... 47 1.5 Biotechnological and Industrial Applications for BEs ............................................ 51 1.6 Objectives of the Study ........................................................................................... 57 Chapter 2 - Production of Functional Recombinant Glycogen Branching Enzymes from Deinococcus radiodurans, Thermus Thermophilus and E. coli .................................... 59 2.1 Introduction ............................................................................................................. 59 2.2 Methods ................................................................................................................... 65 2.2.1 Polymerase Chain Reaction (PCR) – Amplification of GBE cDNAs .................. 65 2.2.2 Agarose Gel Electrophoresis ................................................................................ 68 2.2.3 Cloning of GBE cDNAs ....................................................................................... 68 2.2.4 Expression of Recombinant GBEs in BL21 and ArcticExpress E. coli Strains ... 72 2.2.5 Recombinant Protein Purification ........................................................................ 75 v 2.2.6 Protein Concentration Determination ................................................................... 78 2.2.7 Sodium-Dodecyl-Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) .... 79 2.2.8 Coomassie Blue Protein Staining ......................................................................... 79 2.2.9 Immunodetection of Proteins - Western Blotting ................................................. 80 2.2.10 GBE Zymogram Assay ...................................................................................... 81 2.2.11 Mass Spectrometry ............................................................................................. 81 2.3 Results ..................................................................................................................... 83 2.3.1 Expression and Purification of Recombinant GBEs in BL21 and ArcticExpress Cells ............................................................................................................................... 83 2.3.1.1 EcGBE Expressed from pET28a and pTXB1 Vectors ...................................... 84 2.3.1.2 TtGBE, DrGBE, AaGBE and DgGBE Expressed from pET28a ...................... 87 2.3.1.3 TtGBE, DrGBE, AaGBE and DgGBE Expressed from pTXB1 ....................... 90 2.3.2 Purification of Recombinant GBEs from E. coli .................................................. 92 2.3.3 Determination of Catalytic Activity of Purified GBEs by Zymogram Analysis . 97 2.4 Discussion ............................................................................................................... 99 Chapter 3 - BE Thermal Stability and Chimeric Protein Design, Expression, and Purification .................................................................................................................. 103 3.1 Introduction ........................................................................................................... 103 3.2 Methods ................................................................................................................. 104 3.2.1 Circular Dichroism (CD) Spectroscopy for Protein Folding Clues ................... 104 3.2.2 Chimeric Branching Enzyme Constructs, Expression, Purification, and Analysis of Activity and Products .............................................................................................. 106 3.2.3 Iodine-Binding Assay for Detection of Recombinant BE Activity .................... 107 3.2.4 Analysis of Products of BE Reactions – HPAEC .............................................. 108 3.3 Results and Discussion .......................................................................................... 109 3.3.1 CD Spectroscopy for Assessing
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