Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis:
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| Format: | Abschlussarbeit Elektronisch Software E-Book |
| Sprache: | English |
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Leipzig
[2017]
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| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | 1 CD-ROM (156 Seiten) Illustrationen 12 cm |
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| 100 | 1 | |a Zernia, Sarah |d 1988- |e Verfasser |0 (DE-588)1152826670 |4 aut | |
| 245 | 1 | 0 | |a Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis |c vorgelegt von Master of Science Sarah Zernia |
| 264 | 1 | |a Leipzig |c [2017] | |
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| 502 | |b Dissertation |c Universität Leipzig |d 2017 | ||
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
TABLE OF
CONTENTS.........................................................................................5
ABBREVIATIONS....................................................................................................9
BIBLIOGRAPHISCHE DARSTELLUNG
.........................................................
10
ZUSAMMENFASSUNG.........................................................................................
14
SUMMARY...............................................................................................................
20
CHAPTER I - TUNING CYTOCHROME P450 BM3 FOR APPLICABILITY IN CHEMICAL
INDUSTRY
1.1
ABSTRACT...................................................................................................................
26
1.2 BIOCATALYSTS FOR ORGANIC
SYNTHESIS..........................................................................27
1.3 THE CYTOCHROME P450
FAMILY................................................................................
28
1.4 CYTOCHROME P450 BM3
(CYP102A1)..................................................................
32
1.4.1 P450 BM3 STRUCTURE * UNDERSTANDING THE ENZYME
........................................
33
1.4.2 P450 BM3 ENGINEERING - OPTIMIZING THE ENZYME
..........................................
36
1.4.3 P450 BM3 IMMOBILIZATION * INDUSTRIALIZING THE
ENZYME...............................38
1.4.4 P450 BM3 COFACTOR REGENERATION AND BYPASSING * ELECTRIFYING THE
ENZYME. 39
1.5
OBJECTIVES...............................................................................................................
41
1.6
ACKNOWLEDGMENTS...................................................................................................42
1.7
REFERENCES...............................................................................................................
42
CHAPTER II - PEPTIDE-MEDIATED SPECIFIC IMMOBILIZATION OF CATALYTICALLY
ACTIVE CYTOCHROME P450 BM3 VARIANT
2.1
ABSTRACT...................................................................................................................
54
2.2
INTRODUCTION............................................................................................................
54
2.3 EXPERIMENTAL
PROCEDURES.......................................................................................
56
2.3.1 CLONING OF 3M-P450BM3
...............................................................................
56
2.3.2 EXPRESSION AND PURIFICATION OF CYTOCHROME P450 BM3 CONSTRUCTS
.............
56
2.3.3 SOLID PHASE PEPTIDE SYNTHESIS O F HIGHSP-BP AND CYS-BP
............................
57
2.3.4 EXPRESSED PROTEIN LIGATION
..............................................................................
58
2.3.5 ITO AND GOLD SURFACE PRODUCTION IN 96-WELL FORMAT
.....................................
58
2.3.6
ELISA...............................................................................................................
59
2.3.7 SCANNING ELECTRON MICROSCOPY AND PROTEIN QUANTIFICATION
............................
59
2.3.8 BROD ACTIVITY ASSAY IN
SOLUTION......................................................................
60
2.3.9 BROD ACTIVITY ASSAY ON
SURFACE......................................................................
60
2.4 RESULTS AND
DISCUSSION...........................................................................................61
2.4.1 SYNTHESIS AND CHARACTERIZATION OF BINDING
PEPTIDES.....................................61
2.4.2 PURIFICATION, LIGATION AND CHARACTERIZATION O F 3M-P450BM3
......................
63
2.4.3 BINDING ASSAY AND PROTEIN QUANTIFICATION ON METAL
SURFACES..........................65
2.4.4 ACTIVITY ASSAY IN SOLUTION AND ON
SURFACE........................................................68
2.4.5
CONCLUSION.........................................................................................................
69
2.5
ACKNOWLEDGEMENT...................................................................................................
69
2.6
REFERENCES................................................................................................................70
2.7
APPENDIX.................................................................................................................
75
2.7.1
INTRODUCTION......................................................................................................
75
2.7.2 EXPERIMENTAL
PROCEDURES.................................................................................75
2.7.3
RESULTS................................................................................................................76
2.7.4
CONCLUSION.........................................................................................................80
2.7.5
REFERENCES.........................................................................................................81
CHAPTER III - SURFACE-BINDING PEPTIDE FACILITATES ELECTRICITY-DRIVEN
NADPH-FREE CYTOCHROME P450 CATALYSIS
3.1 A
BSTRACT....................................................................................................................83
3.2
INTRODUCTION.............................................................................................................83
3.3 RESULTS AND DISCUSSION
...........................................................................................
85
3.4
ACKNOWLEDGEMENTS.................................................................................................
89
3.5
REFERENCES................................................................................................................89
3.6 SUPPLEMENTARY INFORMATION
...................................................................................
92
3.6.1 EXPERIMENTAL
PROCEDURES.................................................................................92
3.6.2 EXPRESSION, PURIFICATION AND CHARACTERIZATION O F FLG-P450 BM3
VARIANTS.95
3.6.3 ACTIVITY ASSAY O F FLAG-P450BM3 CONSTRUCTS
..............................................
98
3.6.4 STRUCTURE PREDICTION FOR IMMOBILIZATION OF RATIONALLY DESIGNED
P450 BM3 .99
3.6.5 QUANTIFICATION O F IMMOBILIZED ENZYME ON ITO
SURFACE..............................101
3.6.6 INFLUENCES ON SURFACE INTEGRITY AND PROTEIN STABILITY
....................................
101
3.6.7
REFERENCES.......................................................................................................
105
CHAPTER IV - CHARACTERIZATION OF SEPARATED P450 BM3 DOMAINS ON METAL
OXIDE BINDING AND MONOOXYGENASE ACTIVITY
4.1
ABSTRACT.................................................................................................................
107
4.2
INTRODUCTION..........................................................................................................
107
4.3 EXPERIMENTAL
PROCEDURES.....................................................................................
109
4.3.1 CLONING O F SINGLE P450 BM3
DOMAINS...........................................................109
4.3.2 EXPRESSION AND
PURIFICATION...........................................................................110
4.3.3 CO DIFFERENCE
SPECTRUM.................................................................................110
4.3.4 TRYPTIC DIGESTION AND MASS
SPECTROMETRY.....................................................110
4.3.5 BINDING STUDIES WITH
ELISA..........................................................................110
4.3.6 7-ETHOXYCOUMARIN ACTIVITY
ASSAY....................................................................
I L L
4.4
RESULTS....................................................................................................................
I L L
4.4.1 EXPRESSION AND ANALYSIS O F SEPARATED P450 BM3 DOMAINS
.........................
I L L
4.4.2 P450 IDENTITY O F HEME-CONTAINING DOMAINS
.................................................
113
4.4.3 BINDING STUDIES ON INDIUM TIN OXIDE
.............................................................
113
4.4.4 ACTIVITY STUDIES OF SEPARATED
DOMAINS..........................................................114
4.5
DISCUSSION.............................................................................................................
115
4.6
ACKNOWLEDGMENTS.................................................................................................117
4.7
REFERENCES.............................................................................................................
117
CHAPTER V - APPLICATION OF P450 BM3 ELECTRODE FOR HYDROXYLATION AND
EPOXIDATION ON INDUSTRIAL RELEVANT SUBSTRATES
5.1
ABSTRACT.................................................................................................................
122
5.2
INTRODUCTION..........................................................................................................
122
5.3 EXPERIMENTAL
PROCEDURES.....................................................................................
125
5.3.1 CLONING OF P450 BM3 M
UTANTS.....................................................................
125
5.3.2 PROTEIN EXPRESSION AND PURIFICATION
..............................................................
126
5.3.3 IDENTITY VERIFICATION BY CO DIFFERENCE SPECTRUM
.........................................
126
5.3.4
ELISA.............................................................................................................
126
5.3.5 NARINGENIN
ASSAY.............................................................................................
126
5.3.6 ENZYMATIC CONVERSION OF STYRENE
..................................................................
127
5.3.7 HYDROGEN PEROXIDE PRODUCTION ASSAY
...........................................................
127
5.3.8 CYCLOOCTAN
ASSAY.............................................................................................
128
5.4
RESULTS....................................................................................................................
128
5.4.1 EXPRESSION AND PURIFICATION OF FOUR DIFFERENT VARIANTS OF
HSP-TAGGED
P450
BM3.................................................................................................................128
5.4.2 ENHANCING OF FLAVONOID SOLUBILITY BY ENZYMATIC HYDROXYLATION
..................
130
5.4.3 EPOXIDATION OF STYRENE AS INTERMEDIATE FOR SYNTHESIS OF FINE
CHEMICALS
....
131
5.4.4 HYDROGEN PEROXIDE SHUNT PATHWAY ON P450 BM3 ELECTRODE
......................
132
5.4.5 P450 BM3 ELECTRODE FOR HYDROXYLATION OF SYNTHETIC POLYMER
PRECURSOR
CYCLOOCTANE...............................................................................................................134
5.5
DISCUSSION..............................................................................................................135
5.6
ACKNOWLEDGMENTS.................................................................................................
139
5.7.
REFERENCES.............................................................................................................139
PUBLICATIONS AND PRESENTATIONS
....................................................
144
CURRICULUM
VITAE.........................................................................................146
SELBSTSTAENDIGKEITSERKLAERUNG...........................................................147
DANKSAGUNG......................................................................................................148
AUTHORS CONTRIBUTIONS
STATEMENT..............................................150
|
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| author | Zernia, Sarah 1988- |
| author_GND | (DE-588)1152826670 |
| author_facet | Zernia, Sarah 1988- |
| author_role | aut |
| author_sort | Zernia, Sarah 1988- |
| author_variant | s z sz |
| building | Verbundindex |
| bvnumber | BV046138102 |
| ctrlnum | (OCoLC)1119090198 (DE-599)DNB1152389866 |
| discipline | Biologie |
| format | Thesis Electronic Software eBook |
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| spelling | Zernia, Sarah 1988- Verfasser (DE-588)1152826670 aut Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis vorgelegt von Master of Science Sarah Zernia Leipzig [2017] 1 CD-ROM (156 Seiten) Illustrationen 12 cm txt rdacontent c rdamedia cd rdacarrier Dissertation Universität Leipzig 2017 (DE-588)4113937-9 Hochschulschrift gnd-content (DE-588)4139307-7 CD-ROM gnd-carrier B:DE-101 application/pdf http://d-nb.info/1152389866/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031518403&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis Textdatei PDF |
| spellingShingle | Zernia, Sarah 1988- Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis |
| subject_GND | (DE-588)4113937-9 (DE-588)4139307-7 |
| title | Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis |
| title_auth | Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis |
| title_exact_search | Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis |
| title_full | Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis vorgelegt von Master of Science Sarah Zernia |
| title_fullStr | Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis vorgelegt von Master of Science Sarah Zernia |
| title_full_unstemmed | Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis vorgelegt von Master of Science Sarah Zernia |
| title_short | Peptide-mediated immobilization of cytochrome P450 BM3 for electrocatalysis |
| title_sort | peptide mediated immobilization of cytochrome p450 bm3 for electrocatalysis |
| topic_facet | Hochschulschrift CD-ROM |
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| work_keys_str_mv | AT zerniasarah peptidemediatedimmobilizationofcytochromep450bm3forelectrocatalysis |
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