Identification and characterization of gallium-binding peptides

Autor: Schönberger, Nora
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Druh dokumentu: Text<br />Doctoral Thesis
DOI: 10.1016/j.chroma.2019.04.037
Popis: The present work demonstrates how a peptide-based material can be obtained for the biosorptive recovery of metals from contaminated industrial wastewater. Starting with Phage surface display for the initial identification and optimization of gallium-binding peptides, all the following application-focussed experiments are based on chemically synthesized peptides. Two chromatography-based biopanning methods for the identification of gallium-binding peptides from a commercial phage display library were developed. Five gallium-binding peptide sequences were identified and evaluated to show good gallium-binding properties. Furthermore, the biosorption of free gallium and arsenic by gallium-binding bacteriophage clones was investigated. A large influence of the pH-value on the respective interactions was demonstrated. Mutagenesis experiments were also carried out for a bacteriophage clone expressed peptide, in which a cysteine pair systematically replaced amino acids. Biosorption experiments with the resulting seven different bacteriophage mutants suggested a relationship between the rigidity of the peptide structure and the gallium-binding properties. In isothermal titration experiments, the thermodynamics of the interaction between gallium and the peptides as chemically synthesized derivatives were characterized, independent of the bacteriophage. The peptides differed strongly in their interaction with gallium, and in some cases, the complex formation with gallium depended strongly on the surrounding buffer conditions. The peptide with the amino acid sequence NYLPHQSSSPSR has particularly promising gallium-binding properties. Computer modeling suggests the probable structure of the peptide in aqueous solution and postulates a possible binding site for gallium. The side-selective and covalent immobilization of the peptides on a polystyrene matrix led to the creation of a biocomposite for the biosorptive recovery of gallium. The sorption performance and desorbability of the peptide-based biosorption materials were determined in studies with model solutions and real waters from the semiconductor industry. :EIDESSTATTLICHE VERSICHERUNG II SUMMARY 7 CHAPTER I. 8 Utility of biotechnological approaches in resource technology 9 Phage Surface Display for the recovery of inorganic binding peptides 14 Gallium – Example of a high-tech metal 22 Aims and context of the present work 22 CHAPTER II. 25 Author contributions 25 Abstract 25 Introduction 26 Materials and Methods 28 2.1 Phage Display Library system 28 2.2 Biopanning experiments 29 2.3 Single clone identification 31 2.4 Single clone binding studies 31 Results 32 3.1 Immobilization of gallium ions 32 3.2 Biopanning experiments 33 3.3 Single clone binding studies 38 Discussion 39 4.1 Gallium ions as biopanning target 39 4.2 Phage clone selection 40 Conclusion 44 Acknowledgements 45 CHAPTER III. 46 Author contributions 46 Abstract 47 Introduction 47 Materials and Methods 49 2.1 Handling of phage display library clones 49 2.2 Site-directed mutagenesis experiments 50 2.3 Biosorption experiments 51 Results and Discussion 52 3.1 Experimental context 52 3.2 Original phage clone characterization 53 3.3 Site-directed mutagenesis experiments 56 3.4 Mutant phage clone characterization 57 Conclusions 59 Acknowledgements 60 CHAPTER IV. 61 Author contributions 61 Textual and graphical abstract 62 Introduction 63 Methods 65 2.1 Peptides 65 2.2 Isothermal titration microcalorimetry (ITC) 65 2.3 Preparation of peptide conjugates 65 2.4 Biosorption studies 66 2.5 Model calculation of peptide C3.8 67 Results and Discussion 68 3.1 Interaction studies of free peptides in solution 68 3.2 Biosorption studies with peptide polystyrene conjugates 71 3.2.1 Covalent and site-selective immobilization of peptides 71 3.2.2 Interaction of peptide conjugates with gallium 72 3.2.3 Interaction of peptide conjugates with arsenic 73 3.2.4 Continuous experiments 73 3.3 Model calculation for peptide C3.15 75 Counclusion 76 Acknowledgment 77 CHAPTER V. 78 Obtained insights for the selection of metal-binding peptides in biopanning experiments 79 Conclusions for the development of peptide-based materials for the biosorptive recovery of metal ions from aqueous solutions 81 REFERENCES 85 APPENDIX 94 SUPPORTING INFORMATION FOR CHAPTER IV 94 LIST OF FIGURES 99 LIST OF TABLES 100 LIST OF ABBREVIATIONS 101 LIST OF CHEMICALS 104 ACKNOWLEDGEMENTS 106 CURRICULUM VITAE 109 LIST OF PUBLICATIONS 111
Databáze: Networked Digital Library of Theses & Dissertations
Externí odkaz: