TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Bioverfahrenstechnik Surface Functionalization of Nano-scale Membrane Reactors for Multienzyme Syntheses Ludwig Johann Klermund, M.Sc. Vollständiger Abdruck der von der Fakultät für Maschinenwesen der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr.-Ing. Dirk Weuster-Botz Prüfer der Dissertation: 1. TUM Junior Fellow Dr. rer. nat. Kathrin Castiglione 2. Univ.-Prof. Dr. rer. nat. habil. Dieter Langosch Die Dissertation wurde am 15.11.2016 bei der Technischen Universität München eingereicht und durch die Fakultät für Maschinenwesen am 22.02.2017 angenommen. Acknowledgments This doctoral thesis was prepared at the Institute of Biochemical Engineering (Prof. Dr.-Ing. Dirk Weuster-Botz) of the Technical University of Munich as part of the junior research group of Dr. Kathrin Castiglione. It is a distinct pleasure to thank the many people who contributed to this work. My sincerest gratitude goes to Dr. Kathrin Castiglione for her guidance and outstanding support. I would like to thank her for four fantastic years I spent in her junior research group, for many lively discussions and the vast knowledge she shared. I would like to thank Prof. Dr. Dieter Langosch for his role as committee member and Prof. Dr.-Ing. Dirk Weuster-Botz for his role as chairman. Furthermore, I would like to thank Prof. Dr.-Ing. Dirk Weuster-Botz for his excellent support and for providing the necessary equipment and lab space to work on this thesis. I thank the the BMBF (German Federal Ministry of Education and Research) for funding. A special thanks goes to all of my current and former colleagues, most of all Sarah Poschenrieder, Tom Schwarzer and Florian Sedlmaier, for all the discussions and debates and the excellent collaboration. I would also like to thank Dr. Dirk Hebel for introducing me to the Institute of Biochemical Engineering. I deeply acknowledge my students for their great experimental assistance, especially Miguel Valderrama, Hannah Rosner, Bettina Frank, Simone Gruber, Annique Hunger, Caroline Weinzierl, Tom Wyrobnik, Johannes Müller, Lena Dübbel, Marita Deuschle, Arabella Essert, Gerassimos Kolaitis, Julia Keim, and Jennifer Herrmann. Furthermore, I would like to thank Ellen Truxius, Gabriele Herbrick, Markus Amann, Georg Kojro and Norbert Werth for their support in administrative and technical issues. Last and most important, I would like to thank my family, most of all my wife and little M.K., for all the support and love. i Table of content Table of content ........................................................................................................................ ii 1. Introduction ....................................................................................................................... 1 2. Motivation and objectives .................................................................................................. 3 3. Theoretical background ..................................................................................................... 9 3.1 Polymer vesicles ........................................................................................................ 9 3.1.1 Amphiphilic block copolymers ........................................................................... 11 3.1.2 Polymersome applications ................................................................................ 13 3.1.3 Surface functionalization of polymersomes ...................................................... 16 3.2 Membrane proteins .................................................................................................. 19 3.2.1. Membrane transporters and outer membrane porin OmpF .............................. 20 3.2.2. Membrane associated proteins ......................................................................... 21 3.2.3. Artificial membrane anchoring .......................................................................... 23 3.3 Biocatalysis .............................................................................................................. 24 3.3.1. Enzyme immobilization ..................................................................................... 25 3.3.2. Kinetic parameters of enzymes ........................................................................ 26 3.3.3. Enzyme stability ................................................................................................ 29 3.3.4. Multienzyme syntheses .................................................................................... 30 3.4 Synthesis of cytidine-5’-monophospho-N-acetylneuraminic acid ............................. 32 3.4.1 N-Acyl-D-glucosamine 2-epimerase.................................................................. 34 3.4.2 N-Acetylneuraminate lyase ............................................................................... 36 3.4.3 CMP-sialic acid synthetase ............................................................................... 36 3.5 Green fluorescent protein ........................................................................................ 37 3.6 In vitro protein synthesis .......................................................................................... 38 4. Materials and methods .................................................................................................... 41 4.1 Materials .................................................................................................................. 41 ii 4.1.1 Chemicals and equipment ................................................................................ 41 4.1.2 Biological materials ........................................................................................... 41 4.2 Molecular cloning ..................................................................................................... 41 4.2.1 Polymerase chain reaction ............................................................................... 41 4.2.2 Isolation of plasmid DNA from Escherichia coli ................................................ 42 4.2.3 Agarose gel electrophoresis ............................................................................. 42 4.2.4 DNA purification ................................................................................................ 42 4.2.5 Restriction and ligation of DNA ......................................................................... 42 4.2.6 Site-directed mutagenesis ................................................................................ 43 4.2.7 Preparation of CaCl2-competent cells ............................................................... 43 4.2.8 Transformation of competent cells .................................................................... 43 4.2.9 Colony polymerase chain reaction .................................................................... 44 4.2.10 DNA sequencing ............................................................................................... 44 4.2.11 Molecular cloning of the fusion proteins ........................................................... 44 4.2.12 Cloning of the MBP-TEV-PolyAL-eGFP fusion protein ..................................... 45 4.3 Microbiological methods .......................................................................................... 45 4.3.1 Strain maintenance ........................................................................................... 45 4.3.2 Precultures for heterologous protein expression .............................................. 46 4.3.3 Heterologous expression of fusion proteins ...................................................... 46 4.3.4 Heterologous expression of enzymes ............................................................... 46 4.3.5 Heterologous expression of N-acyl-D-glucosamine 2-epimerase K160I ........... 46 4.3.6 Heterologous expression of membrane channel proteins ................................ 47 4.3.7 Determination of optical density ........................................................................ 47 4.3.8 Cell lysis ............................................................................................................ 47 4.4 Protein purification ................................................................................................... 47 4.4.1 Membrane solubilization ................................................................................... 47 4.4.2 Immobilized metal affinity chromatography ...................................................... 48 4.4.3 Dialysis ............................................................................................................. 48 4.4.4 Hydrophobic interaction chromatography ......................................................... 48 4.4.5 Anionic exchange chromatography .................................................................. 49 iii 4.4.6 Storage of proteins ........................................................................................... 49 4.4.7 Sodium dodecyl sulfate polyacrylamide gel electrophoresis ............................ 49 4.4.8 Determination of protein concentration ............................................................. 50 4.5 Polymersome production and characterization ........................................................ 50 4.5.1 Polymersome preparation ................................................................................. 50 4.5.2 Dynamic light