Analysis of factors influencing enzyme activity and stability in the solid state [Elektronische Ressource] / vorgelegr von Liliya Kulishova

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Analysis of factors influencing enzyme activity and stability in the solid state Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Liliya Kulishova aus Pavlodar Düsseldorf, April 2010 Aus dem Institut für Molekulare Enzymtechnologie (IMET) der Heinrich-Heine Universität Düsseldorf im Forschungszentrum Jülich Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Frau Prof. Dr. Martina Pohl Koreferent: Herr Prof. Dr. Karl-Erich Jäger Tag der mündlichen Prüfung: ii Die vorliegende Arbeit wurde in der Zeit von September 2006 bis September 2009 am Institut für Molekulare Enzymtechnologie der Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich unter der Anleitung von Frau Prof. Dr. Martina Pohl und Dr. Antje Spiess angefertigt. Die Arbeit wurde von der Deutschen Forschungsgemeinschaft im Rahmen des Graduiertenkollegs 1166 "Biocatalysis in non-conventional media (BioNoCo)" gefördert. Diese Dissertation wird u.a. in elektronischer Form auf dem WWW-Server der Universitätsund Landesbibliothek Düsseldorf veröffentlicht.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 65
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Source : DOCSERV.UNI-DUESSELDORF.DE/SERVLETS/DERIVATESERVLET/DERIVATE-16498/DOCTORAL%20THESIS_LILIYA%20KULISHOVA.PDF
Nombre de pages : 169
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Analysis of factors influencing enzyme
activity and stability in the solid state







Inaugural-Dissertation



zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf



vorgelegt von
Liliya Kulishova
aus Pavlodar


Düsseldorf, April 2010

Aus dem Institut für Molekulare Enzymtechnologie (IMET)
der Heinrich-Heine Universität Düsseldorf
im Forschungszentrum Jülich


















Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf




Referent: Frau Prof. Dr. Martina Pohl
Koreferent: Herr Prof. Dr. Karl-Erich Jäger

Tag der mündlichen Prüfung:

ii
Die vorliegende Arbeit wurde in der Zeit von September 2006 bis September 2009
am Institut für Molekulare Enzymtechnologie der Heinrich-Heine-Universität
Düsseldorf im Forschungszentrum Jülich unter der Anleitung von Frau Prof. Dr.
Martina Pohl und Dr. Antje Spiess angefertigt.
Die Arbeit wurde von der Deutschen Forschungsgemeinschaft im Rahmen des
Graduiertenkollegs 1166 "Biocatalysis in non-conventional media (BioNoCo)"
gefördert.
Diese Dissertation wird u.a. in elektronischer Form auf dem WWW-Server der
Universitätsund Landesbibliothek Düsseldorf veröffentlicht.
iii
Table of Contents

Abbreviations xiii

Acknowledgment xv

Publications and conference contributions xvi

Abstract xvii

Summary xviii

Zusammenfassung xxi

1 Introduction 1

1.1 Gas/solid biocatalysis 1
1.1.1 Industrial biocatalysis 1
1.1.2 Principles of gas/solid biocatalysis 2
1.1.3 Activity and stability of enzymes in gas/solid reactor 4
1.1.3.1 Temperature 4
1.1.3.2 Influence of water 5
1.1.3.3 Thermodynamic activity of substrates 7
1.1.3.4 Enzyme preparation 7
1.1.4 State of the art of gas/solid biotransformations with 8
isolated enzymes and whole cells
1.1.4.1 Hydrogenases 8
1.1.4.2 Alcohol oxidases 9
1.1.4.3 Lypolitic enzymes 9
1.1.4.4 Thiamine-diphosphate dependent lyases 9
1.1.4.5 Haloalkane dehalogenases 10
1.1.4.6 Alcohol dehydrogenases 11
1.1.4.7 Whole cells 13

1.2 Alcohol dehydrogenases 14
1.2.1 Classification 14
1.2.2 Short-chain ADHs 15
1.2.3 ADH from Lactobacillus brevis 19
iv
1.2.4 ADH from Thermus sp. 23
1.2.5 ADH from Flavobacterium frigidimaris 23

1.3 Nucleotide cofactors 24
1.3.1 Stability of nicotinamide cofactors 25
1.3.2 Regeneration of NAD(P)H 27

1.4 Thermostability 29
1.4.1 Factors contributing to enzyme thermostability 29
1.4.2 Structural deatures of thermostable proteins 31
1.4.3 Engineering of protein thermostability 32
1.4.3.1 Rational design 32
1.4.3.2 Directed evolution 32
1.4.3.3 Semi-rational design 34
1.4.3.4 Computational methods 34
1.4.3.5 B-FIT approach to enhance thermostability 35

2 Objectives of the present project 37

3 Materials and methods 38

3.1 Materials 38
3.1.1 Chemicals 38
3.1.2 Equipment and software 38
3.1.3 Bacterial strains and plasmids 40
3.1.4 Oligonucleotides 41

3.2 Molecular Biological Methods 42
3.2.1 Electrocompetent E. coli cells 42
3.2.2 Chemically competent E. coli cells 42
3.2.3 Cultivation of E. coli cells 43
3.2.4 Heterologous overexpression of proteins in E. coli 44
3.2.4.1 Batch overexpression 44
3.2.4.2 Cultivation of mutant variants in 96-well plates 44
3.2.4.3 High cell density cultivation 45
3.2.5 Extraction of plasmid DNA from E. coli 47
3.2.5.1 Miniprep DNA isolation 47
3.2.5.2 Midiprolation 47
3.2.6 Quick-change PCR 47
v
3.2.6.1 Site-directed mutagenesis 48
3.2.6.2 Randomization of positions 42, 44 and 48 48
3.2.7 Restriction digest 49
3.2.8 Sequencing 49
3.2.9 Agarose gel electrophoresis 49
3.2.10 SDS-PAGE 50

3.3 Chromatographic purification of proteins 51
3.3.1 Ion-exchange purification of LbADH and its variants 51
3.3.2 Ion-exchange purificaTADH 52
3.3.3 Desalting of proteins 52
3.3.4 Lyophilisation 53

3.4 Analytical methods 53
3.4.1 Determination of protein concentration 53
3.4.2 LbADH activity assay 54
3.4.2.1 Photometer based assay 54
3.4.2.2 Microtiter plate based assay 55
3.4.3 TADH activity assay 55
3.4.4 FfADH acticity assay 56
3.4.5 Water activity adjustment 57

3.5 Enzyme characterisation 57
3.5.1 LbADH thermostability studies 57
3.5.1.1 Thermostability in dissolved state 57
3.5.1.2 ostability in solid state 57
3.5.2 High-throughput screening of LbADH G37D variants for 58
enhanced thermostability
3.5.2.1 Screening in dissolved state 58
3.5.2.2 Screening in the solid state 58
3.5.2.3 Validation of the screening system 59
3.5.3 Cofactor thermostability studies 60
3.5.3.1 Thermostability in dissolved state 60
3.5.3.2 ostability in solid state 60
3.5.4 Determination of substrate spectrum 61
3.5.5 Determination of temperature optimum 61
3.5.6 Determination of pH optimum 61
3.5.7 Determination of kinetic parameters 62

vi
3.6 Spectroscopic studies of enzyme thermostability 62
3.6.1 Tryptophane fluorescence spectroscopy 62
3.6.1.1 Theoretical principles of the tryptophan fluorescence 62
spectroscopy
3.6.1.2 Experimental setup 65
3.6.2 CD spectroscopy 66
3.6.2.1 Theoretical principles of the CD spectroscopy 66
3.6.2.2 Experimental setup 67
3.6.3 Static light scattering spectroscopy 68
3.6.3.1 Theoretical principles of the static light scattering 68
spectroscopy
3.6.3.2 Experimental setup 69

3.7 Analytical procedures for separation and analysis of cofactor 70
degradation products
3.7.1 HPLC setup 70
3.7.2 MS setup 70

3.8 Gas/solid reactor experiments 71
3.8.1 Enzyme immobilization on glass beads 72
3.8.2 Gas/solid reactor operation 73

4 Results and Discussion 74

4.1 Comparative investigation of thermal stability of NADH and 74
NADPH
4.1.1 Thermostability of cofactors in aqueous solution 74
4.1.2 ostabactors in dry solid state 76
4.1.3 Spectroscopic analysis and separation of the cofactor 77
degradation products

4.2 Characterisation of LbADHwt and LbADH G37D 83
4.2.1 Thermostability in aqueous solution 83
4.2.2 ostability in solid state 88
4.2.3 PH optimum 89
4.2.4 Substrate spectrum 91

4.3 Inactivation mechanisms of the dissolved and solid LbADHwt and 95
LbADH G37D
vii

4.4 Generation of thermostable variants 106
4.4.1 Construction and screening of the LbADH G37D 106
mutant library
4.4.2 Setup and validation of a high throughput screening 107
(HTS) system
4.4.3 Characterization of selected stabilized variants 109
4.4.4 Explanation of stabilising effects on molecular level 114

4.5 Characterisation of novel thermostable alcohol dehydrogenases 117

4.6 Gas/solid reactor experiments 122

5 Conclusions and outlook 130

6 References 132

Appendix 144



viii
Figures

Figure 1-1 Schematic representation of the gas/solid biocatalysis. 3
Figure 1-2 Isoterm adsorption curve. 6
Figure 1-3 ADH catalysed reaction. 14
Figure 1-4 Multiple sequence aligment of several SDR enzymes. 17
Figure 1-5 Proposed catalytic reaction mechanism of SDRs. 18
Figure 1-6 Tertiary and quaternary structure of the wild type LbADH. 20
Figure 1-7 Close-up view of the most important residues of the 21
extended proton relay system.
Figure 1-8 Comparison of the NADP-dependent LbADHwt with the 22
NAD-dependent LbADH G37D.
+Figure 1-9 NAD(P) and NAD(P)H . 24
+ +Figure 1-10 Rate constants for the decomposition of NAD , NADP , 26
NADH and NADPH.
Figure 1-11 Schematic representation of cofactor regeneration 28
strategies.
Figure 1-12 Schematic outline of a typical directed evolution 33
experiment.
Figure 3-1 Jablonski diagram. 63
Figure 3-2 Absorption and emission spectra of aromatic amino acids in 64
water, pH 7.0.
Figure 3-3 Conformational changes and CD spectra of poly-L-Lys in 67
aqueous solution.
Figure 3-4 Schematic representation of a continuous gas/solid reactor. 72
Figure 4-1 Thermal stability of the dissolved NADH and NADPH. 75
Figure 4-2 Half lifes of the dissolved NADH and NADPH. 76
Figure 4-3 Thermal stability of solid NADPH and NADH at 50°C by 77
analysis of absorption at 340 nm and relative activity.
Figure 4-4 Absorption spectra of the nicotinamide cofactors and 78
products of their thermal degradation.
Figure 4-5 Fluorescence emission spectra of the nicotinamide 78
cofactors and products of their thermal degradation.
Figure 4-6 2D-HPLC-Chromatogramms of NADH and NADPH. 79
Figure 4-7. Thermal inactivation of the dissolved LbADHwt and LbADH 84
G37D.
Figure 4-8 Thermal stability of the dissolved LbADHwt and LbADH 84
G37D.
ix
Figure 4-9 Concentration-dependent inactivation of LbADHwt at 55 °C. 86
Figure 4-10 Arrhenius plot of inactivation constants of the dissolved 88
LbADHwt and LbADH G37D.
Figure 4-11 Thermal stability of the solid LbADHwt and LbADH G37D. 89
Figure 4-12 Arrhenius plot of inactivation constants of the solid 90
LbADHwt and LbADH G37D.
Figure 4-13 PH dependence of oxidative and reductive activity of 92
LbADHwt and LbADH G37D.
Figure 4-14 Unfolding of the LbADHwt and the LbADH G37D in urea 96
and GdnCl by fluorescence spectroscopy.
Figure 4-15 Unfolding of LbADHwt and the LbADH G37D in urea and 97
GdnCl by CD spectroscopy.
Figure 4-16 Thermal inactivation and unfolding of the LbADHwt and the 99
LbADH G37D by fluorescence emission spectroscopy.
Figure 4-17 Thermal inactivation and unfolding of the LbADHwt and the 100
LbADH G37D by static light scattering spectroscopy.
Figure 4-18 Thermal inactivation of the solid LbADHwt and LbADH 101
G37D: solubility properties.
Figure 4-19 Temperature dependence of CD spectra of the dissolved 102
and solid LbADHwt and LbADH G37D.
Figure 4-20 Thermal unfolding of LbADHwt and the LbADH G37D: 105
percentage of the initial helical content.
Figure 4-21 Elipticity in the CD spectra of LbADHwt and LbADH 105 222
G37D as a function of temperature during heating from 25
to 80 °C and cooling from 80 to 25 °C.
Figure 4-22 Schematic representation of the HTS system design. 108
Figure 4-23 Thermal inactivation of 42/44/48-randomized LbADH G37D 110
variants after 60 min incubation at different temperatures in
aqueous solution.
Figure 4-24 The residual activity the 42/44/48-randomized LbADH 111
G37D variants after incubation at 45°C for 60 min in
aqueous solution.
Figure 4-25 Thermal inactivation of the 42/44/48-randomized LbADH 112
G37D variants after 60 min incubation at different
temperatures in the solid state.
Figure 4-26 The residual activity of selected 42/44/48-randomized 113
LbADH G37D variants after incubation at 70°C for 60 min in
the solid state.
Figure 4-27 Location of amino acids 37, 42, 44 and 48 in the tertiary 115
x

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