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Fundamental phenomenological description and experimental optimization of gel-stabilized biocatalysts in a two-phase system [Elektronische Ressource] / von Bastien Doum`eche

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157 pages
Lerhstuhl für Biotechnologie RWTH-Aachen Prof. Dr.-Ing. W. Hartmeier Fundamental phenomenological description and experimental optimization of gel-stabilized biocatalysts in a two-phase system Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation von Diplom-Biochemiker Bastien Doumèche aus Paris 2002 This work was realized from the 1st October 1999 to 13th December 2002 in the Department of Biotechnology of the RWTH-Aachen, Germany, under the direction of Prof. Dr.-Ing. W. Hartmeier and was support by the Deutsche Forschung Gemeinschaft (DFG) in the Sonderforschungsbereich 540: “Model-based experimental analysis of kinetic phenomena in fluid multi-phase reactive Systems”. Berichter: Universitätsprofessor Dr.-Ing. Winfried Hartmeier -Ing. Jochen Buchs Universitätsprofessor Dr. Lothar Elling Universitätsprofessor Dr. Hans Grambow Tag der mündlichen Prüfung: 13.12.2002 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. I wish to thank Professor Winfried Hartmeier for the opportunity he gave a young French student to realize this PhD work in his department. I address also my acknowledgments to Professor Jochen Büchs, Professor Lothar Elling and Professor Hans Grambow who accepted to judge this work and evaluate my scientific value.
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Lerhstuhl für Biotechnologie
RWTH-Aachen
Prof. Dr.-Ing. W. Hartmeier
Fundamental phenomenological description and experimental
optimization of gel-stabilized biocatalysts in a two-phase system
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation
von
Diplom-Biochemiker
Bastien Doumèche
aus Paris
2002 This work was realized from the 1st October 1999 to 13th December 2002 in the
Department of Biotechnology of the RWTH-Aachen, Germany, under the
direction of Prof. Dr.-Ing. W. Hartmeier and was support by the Deutsche
Forschung Gemeinschaft (DFG) in the Sonderforschungsbereich 540: “Model-
based experimental analysis of kinetic phenomena in fluid multi-phase reactive
Systems”.
Berichter: Universitätsprofessor Dr.-Ing. Winfried Hartmeier -Ing. Jochen Buchs
Universitätsprofessor Dr. Lothar Elling
Universitätsprofessor Dr. Hans Grambow
Tag der mündlichen Prüfung: 13.12.2002

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar. I wish to thank Professor Winfried Hartmeier for the opportunity he gave a young French
student to realize this PhD work in his department.
I address also my acknowledgments to Professor Jochen Büchs, Professor Lothar Elling and
Professor Hans Grambow who accepted to judge this work and evaluate my scientific value.

I wish also to associate my work to Doctor Marion Ansorge-Schumacher (and Fly) for
dealing with the “not-always easy” SFB meetings and all the help and friendship we
exchanged during my time in Aachen.

Members of the institute should also be thank because of their happiness, their scientific
discussions, and all which is part of the laboratory life. Trying not to forgot anybody, I
thank Stefan, Monika, Peer, Uwe, Brigitta, Alex, Julia, Sonja, Daria, Wolfgang, Anita, Uli,
Kathrin, Susy, Marc, and really specially Regina (our super-Hiwi) and Gisela (our artist) for
their energy and friendship. I also thank Inès Backman-Remi, Martin Zimmermann and
Ulrich Schaffrath for their kind help.

Internet shouldn’t be forgotten for not loosing contact with my friends and family sprayed
all over Europe who supported me during for some bad days. Thanks specially to Frag,
Gouzou, Toratora, Isa, Flo, Acid-Rusty, Dave and my parents, among others (but I should
also cite Albert, Patrick, Nico, JC, Tigrou, M´sieur Riboul, Caro, Whilelm, but I´ll certainly
forgot some). I specially dedicate this work to all of you.

Finally, I thanks the Deutsche Forschung Gemeinschaft (DFG) who supported me
financially for my work in the Sonderforschungsbereich 540: “Model-based experimental
analysis of kinetic phenomena in fluid multi-phase reactive Systems”. CONTENT
1 OUTLINE OF THE THESIS 1
2 THEORETICAL BACKGROUND 3
2.1 Heterogeneous biocatalysis 3
2.1.1 Heterogeneity 4
2.1.2 Water activity 4
2.1.2-A. Thermodynamics aspects 4
2.1.2-B. Macromolecular aspects 8
2.1.2-C Control of water activity 10
2.2 Lipases: Generalities 11
2.2.1 Interfacial activation defines a lipase 11
2.2.2 Three-dimensional structure of lipases 13
2.2.3 Reactional mechanism 13
2.2.4 Lipases in biocatalysis 15
2.3 Specificity of Candida rugosa lipase 17
2.3.1 Molecular biology of Crl 17
2.3.2 Purification and cloning 19
2.3.3 Specificity and applications 19
2.4 Enzyme immobilization 21
2.4.1 Reactivity of amino acids 22
2.4.2 Supports for covalent immobilization 23
2.4.3 Cross-linking 24
2.4.4 Adsorption on solid material 24
2.4.5 Entrapment and encapsulation 25
2.5 Calcium-alginate 27
2.5.1 Alginate structure
2.5.2 Calcium-alginate gel structures and ionotropic gellification 28
2.5.3 Use of alginate in biotechnology 31
3 MATERIAL AND METHODS: 33
3.1 Chemicals 33
3.2 Analytical methods
3.2.1 Gas chromatography 33
- i - 3.2.2 HPLC 34
3.2.3 Titrimetry
3.2.3-A Water content in organic media 34
3.2.3-B Determination of the calcium propionate dissociation constant 35
3.2.4 Atomic absorption spectroscopy 35
3.2.5 Determination of partition coefficients 36
3.3 Protein purification methods 36
3.3.1 Polyethylene glycol precipitation 36
3.3.2 Protein precipitation 36
3.3.3 Chromatographic separation of proteins 37
3.3.3-A Hydrophobic interaction chromatography 37
3.3.3-B Size exclusion chromatography 37
3.3.4 Adsorption of Triton X-100 on solid surfaces 37
3.3.5 Activity localizations : MUF-Butyrate activity test. 37
3.3.6 Ultrafiltration 38
3.3.7 Dialysis
3.3.8 SDS-PAGE electrophoresis 38
3.3.9 Analytic isoelectric focusing (IEF) 39
3.3.10 Silver staining 39
3.3.11 Zymogram 40
3.3.12 Hydrolysis of tributyrine
3.3.13 Protein quantification 40
3.4 Molecular modelling 41
3.5 Immobilization method and Alginate gel characterization
3.5.1 Immobilization in calcium-alginate spheres 41
3.5.1-A Solutions 41
3.5.1-B Beads of 2-4 mm diameter 42
3.5.1-C Beads of 100-200 µm diameter 42
3.5.1-D Alginic acid beads 42
3.5.2 Analysis of beads size 42
3.5.3 Density of alginate beads 43
3.5.4 Determination of changes in bead weight 43
3.5.5 Alginate beads resistance to pressure and breaking point. 44
3.6 Chemical methods 45
3.6.1 Inhibitor synthesis 45
- ii - 1 313.6.2 H- and P-NMR 47
3.6.3 Thin layer chromatography 47
3.6.4 Inhibition by DNPHP 47
3.6.5 Partial phase diagram determination 47
3.6.6 Fluorescent labeling of white egg albumin. 48
3.7 Enzymatic reactors 48
3.6.1 Batch reactor 48
3.6.2 Overhead reactor 48
3.6.3 Fluidized-bed reactor 49
3.6.4 One-bead reactor 49
3.6.5 Micro-reactor 49
4. RESULTS AND DISCUSSION 51
4.1 General characterization of calcium-alginate beads 51
4.1.1 Size and weight 51
4.1.2 Efficiency of the lipase entrapment 54
4.1.3 Diffusion of substrates 55
4.1.4 Esterification reaction 57
4.1.5 Free calcium concentration
4.2: Calcium-alginate beads alteration: the water drop phenomena 58
4.2.1 What is the water drop ? 58
4.2.2 Systematic study of the water drop 58
4.2.3 Chemical analysis of the water drop 61
4.2.4 Resistance of the beads to initial pressure 62
4.2.5 Particle sizing experiements 62
4.2.6 Explanation of the water drop by alginic acid hydrogel formation. 64
4.3 Agglomeration and suspension of 100-200 µm calcium-alginate beads 66
4.3.1 Changing the polarity between the solvent and the alginate 66
4.3.2 Addition of surfactants 70
4.3.2-A Non ionic surfactant 70
4.3.2-B Ionic surfactants 71
4.3.2-C Partial determination of the hexane/water/CTAC phase diagram 75
4.3.3 Alginate bead suspension for fluidized-bed reactor 77
4.4 Purification of Candida rugosa lipase from commercial preparation 81
4.4.1 Polyethylene glycol precipitation 81
- iii - 4.4.2 Hydrophobic Interactions chromatography 82
4.4.2-A Elution by decreasing the polarity 83
4.4.2-B Elution by a detergent 84
4.4.2-C Increase of the chaotropic effect 85
4.4.3 Isoelectric focusing analysis: Silver staining and zymogram 86
4.5 Inhibition of Crl by amphiphilic phosphonate 87
4.5.1 Chemical characterization of DNPHP 87
4.5.2 Inhibition of encapsulated Crl from diverse preparations. 90
4.5.3 Synthesis of PBNPHP 94
4.6 Alginate as ion exchange support for proteins 95
4.6.1 Size exclusion chromatography 96
4.6.2 Fluorescence study with entrapped white egg albumin 97
4.7 Influence of calcium on the system 99
4.7.1 Determination of the calcium propionate dissociation constant 99
4.7.2 Influence of the calcium concentration on the enzyme activity 101
4.7.2-A Effect of the calcium on the content of lipase immobilized 102
4.7.2-B Influence of calcium on partition 104
4.8 Control of the pH 108
4.8.1 pH control with Amines 108
4.8.2 pH Control with high buffer concentration 113
4.8.3 pH control with magnesium hydroxide 113
4.9 pH-controlled system: Molecular understanding of the activity 116
4.9.1 Butylamine effects 116
4.9.2 Ionization state of the propionic acid: effect on partition and activity 119
4.9.3 Molecular explanation of the acidic pH dependence for ester synthesis of Crl 119
4.9.4 Comportment of the alkaline Humicola lanuginosa lipase in the system 122
4.9.5 Inactivation by butylamine 126
4.10: Replacement of a lipase by an esterase 128
5. SUMMARY AND PERSPECTIVES 130
6. BIBLIOGRAPHY 133
7. ANNEXES 146
- iv - Outline of the thesis
1. OUTLINE OF THE THESIS
The previous works of Martin Koch (1998) and Frings et al. (1999) show the possibility of
using polysaccharidic matrices in hydrophobic organic solvent to perform enzymatic
reactions. Theses two-phase systems allow a strong partition of polar substrates in the
aqueous phase and an easy recovery of hydrophobic products in the organic phase. Enzymes
and cofactors, which are insoluble in solvent of high hydrophobicity, remain entrapped
inside the aqueous phase. Using polymeric matrices as aqueous phases facilitate the handling
of the immobilized enzyme but also allow to optimize the aqueous phase for optimal
biocatalysis. In contrary to classical systems involving two (or more) liquid phases, enzymes
are suspected to be protected against deleterious effect of the organic solvent when
entrapped in solid hydrogels.
Despite this knowledge, there is a strong lack of information concerning the phenomena
really occurring in the gel phase, looking to physical effects of substrates on the matrix and
on the enzyme, and concerning the interactions between all chemicals and biological
components, from a systematic and rational approach.
This work tries to answer theses questions, specially looking to the chemistry of a model
system, formerly describe in literature (Hertzberg et al., 1992): Lipase from Candida rugosa is
entrapped in calcium-alginate beads and catalyze the esterification of propionic acid butyl
ester from butanol and propionic acid provided in the hexane phase (as depicted in Fig 1). In
contrary to theses authors, an approach of batch reactors is preferred to packed-bed column
previously described.
The description of the system at the molecular and supra-molecular level is performed as
well as its feasibility in a scaled-up process is investigated.
The synthesis of small hydrophobic esters could also be seen as an alternative natural-like
way of flavor production from classical chemistry, as butanol and propionic could be
obtained fermentation of Clostridium acetobutylicum and Propionibacterium acidipropionici
respectively, leading to aroma molecules suitable for the food industry.
An approach of this system with a mathematical model is realized by the collaborating group
of Prof. J. Büchs (Lehrstuhl für Bioverfahrentechnik, RWTH-Aachen) in order to provide us
data and constants inaccessible by classical chemical analysis and to build a predictive model
of the diffusion-reaction events inside the bead (Heinemann, 2003).
- 1 - Outline of the thesis
The work is performed in parallel with 100-200 µm diameter beads of high surface/volume
ratio in the view of scaling up the system to a reactor and with 2-4 mm diameter beads
easier to handle for the determination of the chemicals and physical properties of the system.


Fig. 1: Scheme of the immobilized Candida rugosa lipase in calcium alginate beads in hexane,
catalysing of the esterification of propionate butyl ester.
- 2 -