Characterization of benzaldehyde lyase and benzoylformate decarboxylase in non-conventional media [Elektronische Ressource] / vorgelegt von Mariya Kokova

Mariya Sevela

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2009
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Characterization of benzaldehyde lyase and
benzoylformate decarboxylase in non-conventional
media


Inaugural-Dissertation

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


vorgelegt von

Mariya Kokova
aus Varna



Düsseldorf, Mai, 2009



aus dem Institut für Molekulare Enzymtechnologie
der Heinrich-Heine Universität Düsseldorf









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




Referent: Prof. Dr. Martina Pohl
Koreferent: Prof. Dr. Jörg Pietruszka
Tag der mündlichen Prüfung: 27 Mai 2009
i

Contents
List of figures .......................................................................................................................................... v
List of tables ........... ix
Abbreviations .......................................................................................................................................... x
I. INTRODUCTION ............................... 1
I.1. Why biocatalysis? ......................................................................................................................... 1
I.2. Biocatalysis in non-conventional media ....................... 1
I.2.1. Types of non-conventional media .......................................................................................... 3
I.2.1.1. Organic solvents .............................................. 3
I.2.1.2. Ionic liquids ..................................................................................... 4
I.2.1.3. Supercritical fluids .......... 6
I.2.2. Important factors in non-conventional biocatalysis ............................................................... 7
I.2.2.1. Log P-value ..................................................................................... 7
I.2.2.2. Water activity .................................................. 8
I.2.2.3. “Enzyme memory” .......................................................................... 9
I.2.3. Non-conventional media to solve practical problems of applying benzaldehyde lyase and
benzoylformate decarboxylase ........................................ 9
I.3. Benzaldehyde lyase and benzoylformate decarboxylase ............................................................ 10
I.3.1. Common characteristics ....................................................................... 10
I.3.2. Different main reactions ....................................... 13
I.3.3. Common side reaction – the benzoin synthesis ................................... 14
I.3.4. Mechanistic kinetic model for the benzoin synthesis ........................................................... 17
I.3.5. Structural comparison of BAL and BFD ............................................................................. 18
I.4. Goals ........................................................................... 20
II. MATERIALS AND METHODS...................................................................... 22
II.1. Materials .................................................................... 22
II.1.1. Chemicals ........................................................... 22
II.1.2. Enzymes .............................................................................................. 22
II.1.3. Cultivation media ................................................................................................................ 22 ii

II.2. Microbiological methods ........................................................................................................... 22
II.2.1. Cultivation of BAL and BFDH281A in a shake flask ......................................................... 22
II.2.2. Cultivation of BFDH281A in a fermenter ........................................................................... 23
II.2.3. Cell harvest ......................................................................................................................... 23
II.2.4. Cell desintegration .............. 23
II.3. Molecular biology methods ....................................................................................................... 24
II.3.1. Transformation of chemically competent E. coli cells ....................................................... 24
II.4. Protein chemistry methods......................................................................................................... 24
II.4.1. Enzyme purification ............ 24
II.4.2. Protein determination .......................................................................................................... 25
II.4.3. SDS-PAGE ......................................................................................................................... 25
II.4.4. Determination of enzyme activity. Activity assays ............................ 25
II.4.4.1. Benzoin cleavage assay (BAL only) ............................................................................ 25
II.4.4.2. Benzoylformate decarboxylase assay (BFDH281A only) ........... 26
II.4.4.3. 3,3´,5,5´-tetramethoxy-benzoin formation assay ......................................................... 26
II.4.4.4. Benzoin formation assay by HPLC ............................................. 27
II.4.4.5. Determination of benzoin synthesis in supercritical CO ............................................ 28 2
II.4.5. Determination of enzyme stability ...................................................... 28
II.4.5.1. Determination of thermostability ................................................. 28
II.4.5.2. Determination of enzyme stability in non-conventional media ... 29
II.4.6. Determination of cofactor dissociation ............................................... 30
II.4.7. Determination of enantioselectivity .................................................... 30
II.4.8. Determination of the enzymes spectral properties .............................. 30
II.4.8.1. Tryptophan fluorescence spectroscopy ........................................................................ 30
II.4.8.2. Circular dichroism spectroscopy ................. 31
II.4.9. Determination of pH in cosolvent systems ......... 32
II.4.10. Determination of benzaldehyde and benzoin solubility .................................................... 32
II.5. Reaction kinetics and mechanism .............................................................. 32
II.5.1. Macro kinetics by initial rate measurements ...................................................................... 32
II.5.2. Micro kinetics by progress curve analysis .......... 33 iii

1II.5.3. H NMR spectroscopy. Qualitative analysis of reaction intermediates .............................. 34
III. RESULTS AND DISCUSSION...................................................................................................... 36
III.1. Activity, stability and spectral properties of BAL and BFDH281A ........ 36
III.1.1. Effect of water-miscible organic solvents ......................................................................... 36
III.1.1.1. DMSO as a cosolvent. Effect of pH and temperature ................ 36
III.1.1.1a. Enzyme activity in DMSO/aqueous buffer mixtures ............................................ 37
III.1.1.1b. Enzyme stability in DMSO/aqueous buffer mixtures ........... 41
III.1.1.2. Acetone, ethanol and 2-propanol as cosolvents .......................................................... 45
III.1.2. Effect of water-miscible ionic liquids ............................................... 49
III.1.2.1. Ecoeng 21M as a cosolvent ........................................................ 50
III.1.2.1a. Enzyme activity in Ecoeng 21M/aqueous buffer mixtures ................................... 50
III.1.2.1b. Enzyme stability in Ecoeng 21M/aqueous buffer mixtures .. 54
III.1.2.2. Ecoeng 1111P as a cosolvent ..................................................................................... 56
III.1.2.3. Other ionic liquids ...................................................................................................... 57
III.1.2.4. Hofmeister series of ionic liquids ............... 59
III.1.3. Studies on enzyme conformation in presence of organic solvents and ionic liquids ........ 60
III.1.3.1. Investigation of enzyme unfolding by tryptophan fluorescence spectroscopy ........... 60
III.1.3.2. Investigation of the helical content by CD spectroscopy ........................................... 64
III.2. Activity and stability of BAL and BFDH281A in water-free media ........ 66
III.2.1. Effect of pure organic solvents and ionic liquids .............................................................. 66
III.2.1.1. Activity of BAL in pure organic solvents and ionic liquids ....................................... 66
III.2.1.2. Stability of BAL and BFDH281A in pure organic solvents ....... 68
III.2.2. Effect of supercritical carbon dioxide ............................................................................... 70
III.3. Role of cofactor ........................................................ 72