Development of radical-stable peroxidases based on peroxidase inactivation mechanism

학위논문 (박사)-- 서울대학교 대학원 : 협동과정 바이오엔지니어링전공, 2015. 2. 한지숙.Peroxidases catalyze a variety of oxidative transformations of many aromatic compounds and thus have potential in biosynthesis and other biotechnological applications. However, the usefulness of these versatile enzymes is limited, as the enzyme is quickly inactivated during the oxidation reaction of aromatic compounds. This low stability of peroxidases results in low product yield due to the incomplete reaction and increased production costs. Many researchers have studied this, and three possible pathways for peroxidase inactivation have been proposed: reaction with excess hydrogen peroxide, sorption by polymer product, and reaction with radical intermediates. The first two pathways have been corroborated with extensive evidencehowever, the free radical-mediated mechanism of peroxidase inactivation has not been fully elucidated. Thus, the dominant inactivation mechanism in the oxidation reaction of phenolic compounds must be revealed. An understanding of the molecular mechanism of radical-mediated inactivation is necessary for protein engineering to improve peroxidase stability. Firstly, the dominant mechanism of peroxidase inactivation during phenol oxidation was determined. Two peroxidases, Coprinus cinereus peroxidase (CiP) and horseradish peroxidase isozyme C (HRPC), showed much higher inactivation rates after the simultaneous addition of phenol and hydrogen peroxide. After the oxidation reaction of phenol, the molecular weights of polypeptides originating from the inactivated peroxidases were slightly increased, and a large fraction of heme from the two inactivated peroxidases remained intact. These findings support the hypothesis that the inactivation of peroxidase during the oxidation of phenol occurs by the coupling of phenoxyl radicals with peroxidase polypeptides. Secondly, the radical coupling site of CiP was identified, and the radical stability of CiP was improved by site-directed mutagenesis. The residue F230 of CiP modified with the phenoxyl radical was mutated to amino acids (Ala) that resist radical coupling. The F230A mutant showed the highest stability against the radical attack, retaining 80% of its initial activity, while the wild-type protein was almost completely inactivated. In addition, no structural changes were observed in CiP after radical coupling. Thirdly, HRPC was also engineered to enhance the radical stability. Phenylalanine residues that are vulnerable to modification by phenoxyl radicals were identified and then changed to Ala to prevent radical coupling. The F68A/F142A/F143A/F179A mutant exhibited dramatic enhancement of radical stability, retaining 41% of its initial activity compared to the wild type, which was completely inactivated. Radical coupling did not change the secondary structure or the active site structure of HRPC. Structure and sequence alignment revealed that radical-vulnerable Phe residues were conserved in homologous peroxidases. Fourthly, the radical-stable CiP mutant, F230A, was applied to the major practical applications, such as the removal of phenol, the decolorization of dye, and the synthesis of polymers. As expected, the removal efficiency of phenol and the decolorization efficiency of Reactive Black 5 were increased four- and five-fold, respectively, compared with that of the wild type. In addition, the phenolic polymer having the highest molecular mass (8850 Da) was synthesized by the F230A mutant in a 50% v/v isopropanol-buffer mixture. A novel engineering strategy to eliminate the radical coupling site increased the radical stability of two peroxidases, CiP and HRPC. This implies that phenoxyl radicals covalently bind to critical Phe residues and inactivate peroxidase by blocking substrate access to the active site of the enzyme.ABSTRACT i CONTENTS iv LIST OF TABLES x LIST OF FIGURES xii LIST OF ABBREVIATIONS xv CHAPTER 1 INTRODUCTION 1 1. 1 Research Backgrounds 2 1. 2 Research Objectives 5 CHAPTER 2 LITERATURE SURVEY 9 2. 1 Peroxidase 10 2. 1. 1 Heme peroxidase classification 10 2. 1. 2 Catalytic mechanism of peroxidase 11 2. 1. 3 Horseradish peroxidase (HRP) 14 2. 1. 4 Peroxidase from C. cinereus 16 2. 2 Applications of Peroxidases 18 2. 3 Inactivation Mechanism of Peroxidase 24 2. 3. 1 Inactivation by hydrogen peroxide 24 2. 3. 2 Inactivation by reaction product 28 2. 3. 3 Inactivation by free phenoxyl radical 30 2. 4 Improvement of Peroxidase Stability through Protein Engineering 31 CHAPTER 3 PEROXIDASE INACTIVAION BY COVALENT MODIFICATION WITH PHENOXYL RADICAL DURING PHENOL OXIDATION 36 3. 1 Introduction 37 3. 2 Materials and Methods 40 3. 2. 1 Chemicals and reagents 40 3. 2. 2 Enzymes 40 3. 2. 3 Peroxidase stability 41 3. 2. 4 Peroxidase-catalyzed reactions 42 3. 2. 5 SDS-PAGE 42 3. 2. 6 HPLC analysis 43 3. 3 Results and Discussion 44 3. 3. 1 Inactivation factors for peroxidase during the phenol oxidation reaction 44 3. 3. 2 The modification of peroxidase polypeptide 47 3. 3. 3 Heme destruction of peroxidase 50 3. 4 Conclusion 53 CHAPTER 4 DEVELOPMENT OF THE RADICAL-STABLE COPRINUS CINEREUS PEROXDIASE (CIP) BY BLOCKING THE RADICAL ATTACK 54 4. 1 Introduction 55 4. 2 Materials and Methods 58 4. 2. 1 Peroxidase expression, purification, and activity assay 58 4. 2. 2 Mass spectrometry analysis 58 4. 2. 3 Molecular docking simulation 61 4. 2. 4 Turnover capacity and radical stability 62 4. 2. 5 Kinetic parameters 63 4. 2. 6 Spectroscopic analysis 64 4. 3 Results and Discussion 66 4. 3. 1 Inactivation of CiP during phenol oxidation 66 4. 3. 2 Formation of an inactive adduct between F230 and the phenoxyl radicals 68 4. 3. 3 Improving radical stability by engineering F230 mutants 72 4. 3. 4 Kinetic studies. 81 4. 3. 5 Molecular docking simulation 83 4. 3. 6 Structure of CiP after the phenol modification 85 4. 4 Conclusion 91 CHAPTER 5 ENGINEERING A HORSERADISH PEROXIDASE C STABLE TO RADICAL ATTACKS BY MUTATING MUTIPLE RADICAL COUPLING SITES 92 5. 1 Introduction 93 5. 2 Materials and Methods 96 5. 2. 1 Materials 96 5. 2. 2 Expression of recombinant HRPC 96 5. 2. 3 Refolding of inclusion body and purification 97 5. 2. 4 Peroxidase activity assay 98 5. 2. 5 Mass spectrometry analysis 98 5. 2. 6 Spectroscopic analysis of HRPC 99 5. 2. 7 Turnover capacity and radical stability 100 5. 2. 8 Molecular docking simulation 101 5. 2. 9 Protein modeling of horseradish peroxidase isoenzyme A2 102 5. 3 Results and Discussion 104 5. 3. 1 Inactivation of HRPC during the phenol oxidation 104 5. 3. 2 Peptide modification of HRPC by radical attack 106 5. 3. 3 Effect of radical modification on structure of HRPC 112 5. 3. 4 Improving the radical stability of HRPC by site-directed mutagenesis of multiple Phe residues 116 5. 3. 5 Kinetic characterization of HRPC wild-type and mutants 124 5. 3. 6 Molecular docking simulation of HRPC wild-type and quadruple mutant 127 5. 3. 7 Highly conversed Phe residues in homologous peroxidases 130 5. 4 Conclusion 135 CHAPTER 6 IMPROVED PRACTICAL USEFULNESS OF PEROXIDASE FROM COPRINUS CINEREUS BY MUTIAON OF PHE230 136 6. 1. Introduction 137 6. 2. Materials and Methods 140 6. 2. 1 Materials 140 6. 2. 2. Peroxidase 140 6. 2. 3 Peroxidase stability 141 6. 2. 4 Removal of phenol 141 6. 2. 5 Decolorization of RB5 142 6. 2. 6 Phenol polymerization 143 6. 2. 7. Kinetic studies 144 6. 3. Results and Discussion 145 6. 3. 1. Phenol removal form aqueous solution 145 6. 3. 2. Decolorization of Reactive Black 5 149 6. 3. 3. Enzymatic polymerization of phenol 151 6. 3. 4. Effect of organic solvent on enzyme stability 156 6. 3. 5. Enzyme stability during phenol oxidation in solvent mixtures 159 6. 3. 6. Kinetic study 162 6. 4. Conclusion 165 CHAPTER 7 OVERALL DISCUSSIONS AND RECOMMENDATIONS 166 BIBLIOGRAPHIES 172 ABSTRACT IN KOREAN 193Docto