Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
|
| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-260207 Inhaltsverzeichnis |
| Beschreibung: | XVI, 195 S. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes |c vorgelegt von Monika M. Meier |
| 264 | 1 | |c 2012 | |
| 300 | |a XVI, 195 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
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| 502 | |a Regensburg, Univ., Diss., 2012 | ||
| 650 | 0 | 7 | |a Pseudomonas diminuta |0 (DE-588)4428434-2 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Molekulare Evolution |0 (DE-588)4812902-1 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Amidasen |0 (DE-588)4302186-4 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Amidasen |0 (DE-588)4302186-4 |D s |
| 689 | 0 | 1 | |a Pseudomonas diminuta |0 (DE-588)4428434-2 |D s |
| 689 | 0 | 2 | |a Molekulare Evolution |0 (DE-588)4812902-1 |D s |
| 689 | 0 | |C b |5 DE-604 | |
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Datensatz im Suchindex
| _version_ | 1804151454002315264 |
|---|---|
| adam_text | TABLE OF CONTENTS | I
TABLE OF CONTENTS
TABLE OF CONTENTS I
LIST OF FIGURES VI
LIST OF TABLES IX
FORMULA INDEX XI
LIST OF ACRONYMS AND ABBREVIATIONS XII
ABSTRACT 1
ZUSAMMENFASSUNG 4
1 INTRODUCTION 7
1.1 ENZYME PROMISCUITY 7
1.2 ORGANOPHOSPHATE COMPOUNDS AND ORGANOPHOSPHATE HYDROLASES (OPH) 8
1.2.1 ORGANOPHOSPHATE COMPOUNDS 8
1.2.2 ORGANOPHOSPHATE HYDROLASES (OPH) 10
1.3 THE (PA)S-BARREL-FOLD 11
1.4 THE AMIDOHYDROLASE SUPERFAMILY (AHS) 13
1.5 BACTERIAL PHOSPHOTRIESTERASE (PTE) AND PHOSPHOTRIESTERASE-LIKE
LACTONASES
(PLL) 15
1.5.1 BACTERIAL PHOSPHOTRIESTERASE (PTE) 15
1.5.2 DR0930: A PHOSPHOTRIESTERASE-LIKE LACTONASE (PLL) 19
1.6 ENZYME DESIGN: RATIONAL DESIGN VERSUS DIRECTED EVOLUTION 21
1.6.1 RATIONAL DESIGN 22
1.6.2 DIRECTED EVOLUTION 22
1.7 OBJECTIVES AND APPROACH IN GENERAL 23
2 MATERIALS 25
2.1 INSTRUMENTATION 25
2.2 CONSUMABLES 28
2.3 CHEMICALS 30
2.4 KITS 31
2.5 ENZYMES 32
2.6 BACTERIAL STRAINS 32
2.7 VECTORS 35
2.7.1 PET VECTORS 35
2.7.2 PTNA VECTOR 35
2.8 OLIGODEOXYNUCLEOTIDES 36
2.8.1 VECTOR-SPECIFIC AMPLIFICATION AND SEQUENCING PRIMERS 36
2.8.2 AMPLIFICATION AND MUTAGENIC PRIMERS FOR DR0930 36
2.8.3 AMPLIFICATION PRIMERS FOR PTE 45
2.9 LADDERS AND MARKERS 45
2.10 BUFFERS AND SOLUTIONS 45
2.10.1 BUFFERS AND SOLUTIONS FOR WORKING WITH E. COLI 46
2.10.2 BUFFERS AND SOLUTIONS FOR SCREENING AND ENZYME KINETICS 47
2.10.3 BUFFERS AND SOLUTIONS FOR WORKING WITH DNA AND AGAROSE GEL
ELECTROPHORESIS.48
HTTP://D-NB.INFO/1044139315
II | TABLE OF CONTENTS
2.10.4 BUFFERS AND SOLUTIONS FOR WORKING WITH PROTEINS 48
2.10.5 BUFFERS AND SOLUTIONS FOR SDS-PAGE 49
2.11 BACTERIAL GROWTH MEDIA 50
2.12 SOFTWARE 50
3 METHODS 52
3.1 PREPARATION OF INSTRUMENTATION AND SOLUTIONS 52
3.2 MICROBIOLOGICAL METHODS 52
3.2.1 CULTIVATION AND STORAGE OF E. COLI STRAINS 52
3.2.2 PREPARATION OF CHEMICALLY COMPETENT E. COLI CELLS (INOUE ET AL.,
1990) 52
3.2.3 TRANSFORMATION OF CHEMICALLY COMPETENT E. COLI CELLS 53
3.2.4 PREPARATION OF ELECTRO-COMPETENT E. COLI CELLS (DOWER ET AL.,
1988) 53
3.2.5 PREPARATION OF DNA FOR ELECTROPORATION 54
3.2.6 TRANSFORMATION OF ELECTRO-COMPETENT E. COLI CELLS 54
3.2.7 DETERMINATION OF TRANSFORMATION EFFICIENCY 54
3.2.8 IN VITRO ACTIVITY SCREENING 54
3.3 MOLECULAR BIOLOGY METHODS 56
3.3.1 ISOLATION AND PURIFICATION OF PLASMID DNA FROM E. COLI 56
3.3.1.1 ISOLATION OF PLASMID DNA IN ANALYTICAL AMOUNTS
(MINI-PREPARATION) 57
3.3.1.2 ISOLATION OF PLASMID DNA IN PREPARATIVE AMOUNTS
(MIDI/MAXI-PREPARATION) 57
3.3.2 DETERMINATION OF DNA CONCENTRATION 57
3.3.3 AGAROSE GEL ELECTROPHORESIS 58
3.3.3.1 ISOLATION OF DNA FRAGMENTS FROM AGAROSE GELS 58
3.3.4 ENZYMATIC MANIPULATION OF DSDNA 59
3.3.4.1 CLEAVAGE OF DSDNA BY RESTRICTION ENDONUCLEASES 59
3.3.4.2 LIGATION OF DNA FRAGMENTS 59
3.3.5 AMPLIFICATION OF DNA FRAGMENTS BY STANDARD PCR (POLYMERASE CHAIN
REACTION) 60
3.3.6 COLONY PCR 62
3.3.7 PCR METHODS FOR SITE-DIRECTED MUTAGENESIS 62
3.3.7.1 QUIKCHANGE SITE-DIRECTED MUTAGENESIS (QCM) 62
3.3.7.2 PHUSION SITE-DIRECTED MUTAGENESIS KIT 64
3.3.7.3 MEGAPRIMER PCR (SARKAR & SOMMER, 1990) 64
3.3.7.4 OVERLAP EXTENSION PCR (HO ETAL., 1989) 65
3.3.8 IN VITRO RECOMBINATION BY STEP (STAGGERED EXTENSION PROCESS) 66
3.3.9 TRANSPOSON-MEDIATED INSERTION MUTAGENESIS (MGS* KIT) 67
3.3.10 CONSTRUCTION OF A GENE LIBRARY 69
3.3.10.1
DETERMINATION OF THE SIZE OF A GENE LIBRARY 70
3.3.11 DNA SEQUENCING 71
3.4 PROTEIN BIOCHEMISTRY METHODS 72
3.4.1 GENE EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEIN 72
3.4.1.1 GENE EXPRESSION IN ANALYTICAL SCALE 72
3.4.1.2 GENE EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS IN
PREPARATIVE
SCALE 73
3.4.2 PROTEIN PURIFICATION FROM THE SOLUBLE CELL FRACTION 73
3.4.2.1 PROTAMINE SULFATE AND AMMONIUM SULFATE PRECIPITATION 73
3.4.2.2 ION EXCHANGE CHROMATOGRAPHY (IEX) 74
3.4.2.3 PREPARATIVE GEL FILTRATION 77
3.4.2.4 DESALTING OF PROTEIN SOLUTIONS 78
TABLE OF CONTENTS | III
3.4.2.4.1 DIALYSIS OF PROTEIN SOLUTIONS 78
3.4.2.4.2 DESALTING AND BUFFER EXCHANGE BY GE ILLUSTRA NAP* OR PD-10
COLUMNS 78
3.4.2.4.3 DESALTING VIA GE SEPHADEX G25 DESALTING 78
3.4.2.5 CONCENTRATING PROTEIN SOLUTIONS 79
3.4.2.6 STORAGE OF PURIFIED PROTEINS 79
3.5 ANALYTICAL METHODS 79
3.5.1 PROTEIN CONCENTRATION DETERMINATION VIA UV-ABSORPTION SPECTROSCOPY
79
3.5.2 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 80
3.5.3 ICP-MS MEASUREMENTS 81
3.5.4 STEADY-STATE ENZYME KINETICS 81
3.5.4.1 COLORIMETRIC ASSAY FOR OPH ACTIVITY WITH P-NITROPHENOL
SUBSTITUTED OPS 81
3.5.4.2 COUPLED DTNB (ELLMAN S REAGENT) ASSAY 82
3.5.4.3 PH-DEPENDENT COLORIMETRIC ASSAY 82
3.5.4.4 DATA ANALYSIS 83
3.5.5 DIXON PLOT FOR COMPETITIVE INHIBITION 83
3.5.6 DETERMINATION OF STEREOPREFERENCE 84
3.6 PROTEIN CRYSTALLIZATION AND X-RAY STRUCTURE DETERMINATION 85
3.6.1 PROTEIN CRYSTALLIZATION 85
3.6.2 DATA COLLECTION 86
3.6.3 DATA PROCESSING AND STRUCTURE DETERMINATION 86
3.7 ROSETTALIGAND DOCKING 87
3.7.1 PROTEIN RECEPTOR AND COFACTOR PREPARATION FOR DOCKING 87
3.7.2 LIGAND PREPARATION AND LIGAND POSITIONING 88
3.7.3 DOCKING CALCULATIONS AND ANALYSIS OF DOCKING RESULTS 88
4 RESULTS AND DISCUSSION 89
4.1 D. RADIODURANS DR0930: A CLOSE HOMOLOGUE OF P. DIMINUTA PTE 89
4.2 DR0930 AND PTE: PROMISCUOUS AND NATIVE ACTIVITIES 93
4.2.1 HETEROLOGOUS EXPRESSION IN E. COLI AND PURIFICATION OF THE
RECOMBINANT
WILD-TYPE DR0930 AND WILD-TYPE PTE PROTEINS 93
4.2.2 CHEMICAL STRUCTURES OF 6-NONANOIC LACTONE AND OP COMPOUNDS 95
4.2.3 STEADY-STATE KINETIC CHARACTERIZATION OF WILD-TYPE DR0930 AND
WILD-TYPE PTE
FOR NATIVE AND PROMISCUOUS ACTIVITIES 97
4.3 LABORATORY EVOLUTION OF WILD-TYPE DR0930 FOR OPH ACTIVITY 102
4.3.1 RANDOMIZATION OF WILD-TYPE DR0930 BY ERROR-PRONE PCR (EPPCR) 103
4.3.1.1 CHARACTERIZATION OF THE EPPCR LIBRARY 103
4.3.1.2 VERIFICATION OF EPPCR HITS BY IN VITRO ACTIVITY SCREENING WITH
EPXN 105
4.3.1.3 HETEROLOGOUS EXPRESSION, PURIFICATION AND IN VITRO
CHARACTERIZATION OF THE
EPPCR HITS 106
4.3.1.4 METAL-DEPENDENCY OF THE OPH ACTIVITY 107
4.3.1.5 STEADY-STATE KINETIC CHARACTERIZATION OF CO/CO-DR0930_5 FOR THE
HYDROLYSIS
OF OPS 1-7 108
4.3.2 IN VITRO RECOMBINATION OF BENEFICIAL 1
ST
GENERATION VARIANTS BY STEP 110
4.3.2.1 GENERATION AND CHARACTERIZATION OF A PTNA-DR0930-STEP LIBRARY
110
4.3.2.2 IN VITRO ACTIVITY SCREENING OF THE PTNA-DR0930-STEP LIBRARY 111
4.3.2.3 HETEROLOGOUS EXPRESSION, PURIFICATION AND IN VITRO
CHARACTERIZATION OF HITS
ISOLATED FROM THE STEP LIBRARY 113
4.3.3 SIMULTANEOUS HOT SPOT MUTAGENESIS (SHM) 114
4.3.3.1 GENERATION AND CHARACTERIZATION OF A SHM LIBRARY 114
IV I TABLE OF CONTENTS
4.3.3.2 IN VITRO ACTIVITY SCREENING OF THE PTNA-C/R0930-SHM LIBRARY
USING A
COMBINATION OF FILTER PAPER AND 96-WELL BLOCK ACTIVITY SCREEN 116
4.3.3.3 HETEROLOGOUS EXPRESSION, PURIFICATION AND STEADY-STATE ENZYME
KINETIC
CHARACTERIZATION OF HITS FROM THE SHM LIBRARY 118
4.3.3.4 GENERATION AND CHARACTERIZATION OF CONSENSUS VARIANTS 120
4.3.4 SEQUENTIAL SITE-DIRECTED, SITE-SATURATION AND COMBINATORIAL
MUTAGENESIS 122
4.3.4.1 COMBINATORIAL MUTAGENESIS ON TEMPLATE DR0930_5 123
4.3.4.2 SITE-SATURATION MUTAGENESIS AT POSITION TYR28 128
4.3.4.3 COMBINATORIAL AND INSERTION/DELETION SCANNING MUTAGENESIS ON
TEMPLATE
DR0930_81 131
4.3.4.3.1 GENERATION OF INSERTION AND DELETION VARIANTS 131
4.3.4.3.1.1 MULTIPLE AMINO ACID DELETION VARIANTS OF PA-LOOP 3 131
4.3.4.3.1.2 TRANSPOSON-MEDIATED INSERTION SCANNING MUTAGENESIS 131
4.3.4.3.2 SITE-SATURATION MUTAGENESIS OF TRY97 ON TEMPLATE DR0930_81 132
4.3.4.4 MUTAGENESIS ON TEMPLATE DR0930_106 135
4.3.4.4.1 SITE-SATURATION MUTAGENESIS AT POSITION PHE26 135
4.3.4.4.2 COMBINATORIAL SITE-DIRECTED MUTAGENESIS ON TEMPLATE DR0930_106
136
4.3.5 SUMMARY: DESIGN OF A MULTI-EFFICIENT OPH 140
4.4 DETAILED CHARACTERIZATION OF BEST ENGINEERED DR0930 VARIANTS 141
4.4.1 STEADY-STATE KINETIC CHARACTERIZATION FOR THE HYDROLYSIS OF
5-NONANOIC
LACTONE (NATIVE ACTIVITY) 141
4.4.2 STEADY-STATE KINETIC CHARACTERIZATION FOR THE HYDROLYSIS OF DEVX
143
4.4.3 STEREOPREFERENCE ANALYSIS OF WILD-TYPE DR0930 AND DR0930_134 144
4.4.4 STRUCTURAL ANALYSIS OF ENGINEERED DR0930 VARIANTS 149
4.4.4.1 CRYSTALLIZATION AND X-RAY STRUCTURE DETERMINATION OF EVOLVED
DR0930
VARIANTS 149
4.4.4.2 ANALYSIS OF BENEFICIAL MUTATIONS IN ENGINEERED VARIANTS 153
4.4.5 LIGAND DOCKING STUDIES WITH ENGINEERED DR0930 VARIANTS 155
4.4.5.1 RETROPERSPECTIVE LIGAND DOCKING 155
4.4.5.2 DOCKING OF EPXN IN THE ACTIVE SITE OF DR0930_134 157
5 FINAL DISCUSSION 161
5.1 DIVERGENT AND CONVERGENT EVOLUTION OF ENZYMES 161
5.2 D. RADIODURANS DR0930: A PROMISCUOUS OP HYDROLYZING ENZYME 162
5.3 LABORATORY EVOLUTION OF DR0930 FOR OP HYDROLYSIS 163
5.3.1 DESIGN OF AN EFFICIENT AND MULTI-FUNCTIONAL OP HYDROLYZING DR0930
VARIANT... 163
5.3.2 DETAILED CHARACTERIZATION OF ENGINEERED DR0930 VARIANTS 167
5.3.3 COMPARISON OF DESIGN ACHIEVEMENS TO STATE-OF-THE-ART DESIGNS FOR
OPH
ACTIVITY 170
5.4 IMPLICATIONS FOR PROTEIN DESIGN AND EVOLUTION 171
5.5 OUTLOOK 172
6 REFERENCES 173
7 APPENDIX 185
7.1 DNA AND PROTEIN SEQUENCES OF WILD-TYPE DR0930 AND WILD-TYPE PTE 185
7.1.1 DNA AND PROTEIN SEQUENCES OF DEINOCOCCUS RADIODURANS R1 DR0930 185
7.1.2 DNA AND PROTEIN SEQUENCES OF PSEUDOMONAS DIMINUTA PTE 186
7.2 STRUCTURE OF VARIOUS SUBSTRATES FOR PLL ENZYMES AND PTE 187
TABLE OF CONTENTS | V
7.3 DETERMINATION OF THE CONVERSION FACTOR 577 FOR THE PH-DEPENDENT
COLORIMETRIC
ASSAY 188
7.4 DETERMINATION OF THE ENZYME METAL CONTENT BY ICP-MS 189
7.5 SEQUENCE OF MOST ACTIVE DR0930 VARIANTS ISOLATED FROM THE
PTNA-C/R0930-SHM
LIBRARY 190
7.6 DIXON PLOT FOR DETERMINATION OF KJ 191
8 ACKNOWLEDGEMENTS 192
9 EIDESSTATTLICHE ERKLARUNG 194
|
| any_adam_object | 1 |
| author | Meier, Monika M. |
| author_facet | Meier, Monika M. |
| author_role | aut |
| author_sort | Meier, Monika M. |
| author_variant | m m m mm mmm |
| building | Verbundindex |
| bvnumber | BV041366504 |
| classification_rvk | WD 5100 |
| collection | ebook |
| ctrlnum | (OCoLC)862829483 (DE-599)BVBBV041366504 |
| dewey-full | 572.79338 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.79338 |
| dewey-search | 572.79338 |
| dewey-sort | 3572.79338 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| owner_facet | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 |
| physical | XVI, 195 S. Ill., graph. Darst. |
| psigel | ebook |
| publishDate | 2012 |
| publishDateSearch | 2012 |
| publishDateSort | 2012 |
| record_format | marc |
| spelling | Meier, Monika M. Verfasser aut Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes vorgelegt von Monika M. Meier 2012 XVI, 195 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2012 Pseudomonas diminuta (DE-588)4428434-2 gnd rswk-swf Molekulare Evolution (DE-588)4812902-1 gnd rswk-swf Amidasen (DE-588)4302186-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Amidasen (DE-588)4302186-4 s Pseudomonas diminuta (DE-588)4428434-2 s Molekulare Evolution (DE-588)4812902-1 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-260207 http://epub.uni-regensburg.de/26020/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-260207 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026814730&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Meier, Monika M. Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes Pseudomonas diminuta (DE-588)4428434-2 gnd Molekulare Evolution (DE-588)4812902-1 gnd Amidasen (DE-588)4302186-4 gnd |
| subject_GND | (DE-588)4428434-2 (DE-588)4812902-1 (DE-588)4302186-4 (DE-588)4113937-9 |
| title | Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes |
| title_auth | Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes |
| title_exact_search | Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes |
| title_full | Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes vorgelegt von Monika M. Meier |
| title_fullStr | Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes vorgelegt von Monika M. Meier |
| title_full_unstemmed | Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes vorgelegt von Monika M. Meier |
| title_short | Reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes |
| title_sort | reconstructing molecular evolution by changing substrate specificities within the amidohydrolase superfamily of enzymes |
| topic | Pseudomonas diminuta (DE-588)4428434-2 gnd Molekulare Evolution (DE-588)4812902-1 gnd Amidasen (DE-588)4302186-4 gnd |
| topic_facet | Pseudomonas diminuta Molekulare Evolution Amidasen Hochschulschrift |
| url | http://epub.uni-regensburg.de/26020/ https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-260207 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026814730&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT meiermonikam reconstructingmolecularevolutionbychangingsubstratespecificitieswithintheamidohydrolasesuperfamilyofenzymes |