Smart hydrogels based platforms for investigation of biochemical reactions DISSERTATION zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Nidhi C. Dubey geboren am 18.10.1985 in Nashik, India Die Dissertation wurde in der Zeit von Februar 2011 bis März 2015 am Leibniz-Institut für Polymerforschung Dresden e.V. angefertigt Gutachter: Prof. em. Dr. rer. nat. Manfred Stamm Prof. Dr. rer. nat. habil. Karl-Heinz van Pée Eingereicht am: 13 May 2015 Tag der Verteidigung: 20 August 2015 TABLE OF CONTENT 1 Introduction ................................................................................................................. 1 1.1 Motivation ............................................................................................................................................ 1 1.2 Goals ....................................................................................................................................................... 6 1.3 Outline ................................................................................................................................................... 7 2 Fundamentals and Methods .................................................................................... 9 2.1 Smart polymer hydrogel ................................................................................................ 9 2.1.1 Introduction ................................................................................................................................ 9 2.1.2 Types of smart polymers .................................................................................................... 10 2.1.3 Core shell microgel and synthesis ................................................................................... 17 2.2 Biochemical reaction ................................................................................................... 25 2.2.1 Acetyl CoA synthetase .......................................................................................................... 25 2.2.2 Pyruvate kinase and lactate dehydrogenase .............................................................. 28 2.3 Enzyme immobilization .............................................................................................. 31 2.3.1 Introduction .............................................................................................................................. 31 2.3.2 Factors affecting enzyme stability .................................................................................. 35 2.3.3 Michaelis Menten kinetics .................................................................................................. 36 2.4 Characterization techniques ..................................................................................... 39 2.4.1 Dynamic light scattering ..................................................................................................... 39 2.4.2 Zeta potential ........................................................................................................................... 41 2.4.3 Scanning electron microscopy .......................................................................................... 43 2.4.4 UV visible absorbance spectroscopy ............................................................................. 46 2.4.5 Fluorescence spectroscopy ................................................................................................ 48 2.4.6 Circular dichroism ................................................................................................................. 49 2.4.7 Enzyme assays ......................................................................................................................... 51 3 Results and discussions .......................................................................................... 53 3.1 Thermo-responsive microgels synthesis and characterization .................... 53 3.1.1 Introduction .............................................................................................................................. 53 3.1.2 PNIPAm-AEMA core shell microgel preparation ...................................................... 53 3.1.3 PNIPAM-PEI core shell microgel preparation ........................................................... 56 3.1.4 Conclusion ................................................................................................................................. 59 3.1.5 Experimental section ............................................................................................................ 59 3.2 PNIPAm-AEMA core-shell microgels support for Acs ....................................... 63 3.2.1 Introduction .............................................................................................................................. 63 3.2.2 Results and discussion ......................................................................................................... 63 3.2.3 Conclusion ................................................................................................................................. 71 3.2.4 Experimental section ............................................................................................................ 72 3.3 Adsorptions of Acetyl CoA synthetase on PNIPAm-PEI 1 microgels ............ 77 3.3.1 Introduction .............................................................................................................................. 77 3.3.2 Results and discussion ......................................................................................................... 78 3.3.3 Conclusion ................................................................................................................................. 87 3.3.4 Experimental section ............................................................................................................ 88 3.4 Sequential enzymes reactions on thermo-responsive microgels ................. 91 3.4.1 Introduction .............................................................................................................................. 91 3.4.2 Results and discussion ......................................................................................................... 92 3.4.3 Conclusion .............................................................................................................................. 102 3.4.4 Experimental section ......................................................................................................... 103 4 Summary ......................................................................................................................... i 5 Future outlook ............................................................................................................. v 6 Appendix ....................................................................................................................... ix 6.1 Abbreviations ..................................................................................................................................... xi 6.2 List of figures ................................................................................................................................... xiii 6.3 List of tables ..................................................................................................................................... xix 7 Bibliography .............................................................................................................. xxi Acknowledgements ...................................................................................................... xliii 1. Introduction 1 Introduction 1.1 Motivation Principally the life on earth is driven by the cascades of biochemical reactions taking place inside the micro-compartments of the cell in presence of the biocatalyst called ‘enzymes’.1,2 Many of the metabolic reactions are catalyzed via one or more membrane-associated multi-enzyme complexes and not actually by free-floating ‘soluble’ enzymes.3,4 By sitting close to each other enzymes carry out reaction in tandem by micro-channeling the molecules, thus overcoming the diffusion barrier due to viscous interior of the cell.3,5,6 Under physiologically mild conditions the enzymes interplay different class of reactions (amidation, acetylation, esterification, oxidation, hydrogenation, etc.) to fulfill the metabolism.1,7,8 The high specificity, efficiency and green nature of the enzyme catalyzed reaction are the beneficial characteristics for the development of clean processes in synthesis of the chiral building blocks and biologically active compounds.9–12 Pharmaceutical 7 6 21 Biotech R&D Diagnostic 9 Biocatalyst Food and Beverage 12 15 fabric and Houshold Biofuel 6 3 Animal feed 21 Figure 1.1. The chart shows the World enzyme market in 2010 for different sectors (The Freedonia Group Inc.).13,14 1 1.1 Motivation The World enzyme market is growing and is expected to rise to $8 billion in 2015 with annual 6.8 % increase in the demand.14 Figure 1.1 depicts the sectors that employee enzymes for their purposes, and clearly indicates pharmaceutical industries as one of the most prominent user of the enzymes. Straathof and co- worker analyzed 134 different industrial transformations data and presented an overview of the use of different enzyme classes and whole cells for the conversions