Exploration of evolutionary pathways Aspergillus niger epoxide hydrolase [Elektronische Ressource] / vorgelegt von Yosephien Gumulya

Gumulya, Yosephine

Informations

• Publié par: ruhr-universitat_bochum
• Publié le: 01 janvier 2010
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Exploration of evolutionary pathways
Aspergillus niger epoxide hydrolase




Dissertation
Zur Erlangung des Grades eines Doktors der Naturwissenschaften der
Abteilung Chemie der Ruhr-Universität Bochum



vorgelegt von M.Sc.
Yosephine Gumulya
Aus Indonesien



Mülheim an der Ruhr
2010
 
 











Dedicated especially for my beloved parents and sister
 
 



















Referent: Prof. Dr. Manfred T. Reetz
Korreferent: Prof. Dr. Martin Feigel
Tag der mündlichen Prüfung:




 
 
Diese vorliegende Arbeit wurde in der Zeit von April 2007 bis Marz 2010 am Max-Planck-Institut für
Kohlenforschung im Mülheim an der Ruhr unter der Leitung von Herrn Prof. Dr. M. T. Reetz durchgeführt.
Herrn Prof. Dr. M. T. Reetz danke ich herzlich für die interessante und herausfordernde Themenstellung,
die gewähre Freiheit bei der Durchführung der Arbeit und jeder erdenklichen Unterstützung bei fachlichen
und darüber hinausgehenden Fragen.
Ich danke Herrn Prof. Dr. M. Feigel für die Übernahme des Korreferats sowie Herrn Prof. Dr. M. Müller für
sein Auftreten als Nebenfachprüfer.
Ich danke Frau R. Lohmer, Frau E. Enk, und Frau A. Rathofer für die außergewöhnliche Unterstützung
und Hilfe in jeglicher Situation.
Frau R. Barabasch danke ich für ihre Hilfe bei der Literaturarbeit.
Dank an alle Mitgliedern des Arbeitskreises, besonders G. Mehler, P. Wedemann und M. Hermes für die
Hilfsbereitschaft.
Ein besonderer Dank für den Mitarbeitern der HPLC Abteilung. A. Deege danke ich für die schnelle
Entwicklung der Trennungsmethode, Heike und Dina für die schnelle Messung meiner unzähligen Proben
(inklusiv am Wochenende).
Special thanks goes to Despina for all her helps in everything, in science (scientific discussions, giving
Maestro tutorials, updating the literature), in being a good company during the night shifts, and mainly for
her patience in reading and revising this thesis.
I thank Ximo, for his help in science (in helping me establishing the experimental platform, scientific
discussions) but mainly for giving the mental support during my tough initial PhD year.
I thank Dirk, for his companionship especially during my difficult times, for his patience in teaching me
AKTA and mainly for the critical input in thermo kinetics.
I would like to thank Layla and Soni who have helped me a lot during my tough first PhD year, Juan Pablo
for the help in MD simulations, Woyciech for the help in size exclusion chromatography, Zheng for being
the helpful epoxide hydrolase lab-mate, and Hermi, Layla, Carlos, Lore for being good office-mates.
I would like also to thank all the group members, the former and present coworkers in the group. It was
really a great experience to meet and to get to know all of you!!!
And last but not least, my parents and my sister. Their constant support and belief in me throughout all
these tough years are the ones that make me could finish my PhD. Without them, this thesis would not
even exist.
 
 
Table of Content
1. Introduction ............................................................................................................................................ 1
1.1. Epoxide hydrolases ........................................................................................................................... 2
1.2. Epoxide hydrolase-catalyzed reactions ............................................................................................. 2
1.3. Epoxide hydrolase from Aspergillus niger (ANEH) ............................................................................ 3
1.4. Directed evolution of Aspergillus niger epoxide hydrolase (ANEH) .................................................. 4
1.5. Hydrolytic kinetic resolution ............................................................................................................... 8
1.6. Protein thermostability ....................................................................................................................... 9
1.6.1. Thermodynamic stability and kinetic stability ............................................................................. 10
1.6.2. Factors responsible for enhanced thermal stability of proteins .................................................. 10
1.6.3. Methodologies that have been developed to evolve protein for better thermostability ............. 13
1.6.4. B-FIT (B-factor iterative test) ...................................................................................................... 17
1.6.5. Enzyme screening for stability .................................................................................................... 17
1.6.6. Enzyme thermal inactivation ...................................................................................................... 18
1.7. Protein fitness landscape ................................................................................................................ 20
1.8. Epistasis and accessibility of mutational paths ............................................................................... 22
1.9. Oversampling in directed evolution .................................................................................................. 23
1.10. Scope and outline of the thesis ..................................................................................................... 24
2. Saturation Mutagenesis for improving ANEH thermostability ........................................................ 26
2.1. Introduction ...................................................................................................................................... 26
2.2. Results and Discussion .................................................................................................................. 26
2.2.1. Development and validation of screening for improved ANEH thermostable mutants ............ 26
2.2.2. Initial round of iterative saturation mutagenesis ..................................................................... 27
2.3. Experimental .................................................................................................................................. 34
3. Exploration of evolutionary pathways in improving ANEH thermostability ................................... 36
3.1. Introduction ....................................................................................................................................... 36
3.2. Results and Discussion ................................................................................................................... 37
3.2.1. The second and third round of ISM to further increase ANEH thermal stability ................. 37
3.2.2. Influence of the sequential order of library generation ........................................................ 40
 
 
 
3.2.3. The fourth, fifth, and sixth round of ISM .............................................................................. 41
3.2.4. Combining mutations versus ISM approach ....................................................................... 45
3.2.5. Temperature dependence of the specific activity ................................................................ 47
3.2.6. Stability of purified ANEH wild type and thermo mutants ................................................... 49
3.2.7. Amino acid substitutions in thermostable ANEH mutants .................................................. 53
3.3.Experimental .................................................................................................................................... 55
4. Exploration of evolutionary pathways in improving ANEH enantioselectivity .............................. 57
4.1. Introduction ..................................................................................................................................... 57
4.2. Results and Discussion ................................................................................................................... 58
4.2.1. Pathways leading to the highly enantioselective mutants ................................................... 60
4.2.2. The fitness-pathway landscape .......................................................................................... 61
4.2.3. Sequence relationships among the best mutants obtained in each evolutionary pathway 63
4.2.4. Structural difference among the best mutants obtained in each evolutionary pathway ...... 65
4.3. Experimental ....................... 67
5. Combining B-FIT and CAST ................................................................................................................ 68
5.1. Introduction ..................................................................................................................................... 68
5.2. Results and Discussion ...................................................................................................