Development of immobilization and drying methods of enzymes on support particles for enzymatic gas-phase reactions [Elektronische Ressource] / vorgelegt von Archana Harendra Trivedi

Ravi

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Lehrstuhl für Bioverfahrenstechnik
RWTH-Aachen
Prof. Dr.-Ing. Jochen Büchs


DEVELOPMENT OF IMMOBILIZATION AND DRYING
METHODS OF ENZYMES ON SUPPORT PARTICLES
FOR ENZYMATIC GAS-PHASE REACTIONS

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des Akademischen
Grades eines Doktorin der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Master of Science (Technology) - Bioprocess Technology
Archana Harendra Trivedi
aus
Indien
2005









This research work was carried out at Department of Biochemical Engineering of
RWTH-Aachen University, Germany, under the supervision of Prof. Dr.-Ing Jochen
Büchs during October 01, 2002 – March 24, 2005 and was mainly supported by
Deutsche Forschung Gemeinschaft (Graduiertenkollegs 440 “Methods in Asymmetric
Synthesis”).
Examiners:
University Professor Dr.-Ing. Jochen Büchs
University Professor Dr.-Ing. Winfried Hartmeier
University Professor Dr.rer.nat. Walter Leitner
University Professor Dr.rer.nat. Ulrich Klinner

Date of oral examination: March 24, 2005

(Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.)






iiABSTRACT
In this research work, two mesophilic alcohol dehydrogenases namely baker’s yeast
alcohol dehydrogenase (YADH) and Lactobacillus brevis alcohol dehydrogenase
(LBADH) and one thermophilic alcohol dehydrogenase namely Thermoanaerobacter
species alcohol dehydrogenase (ADH T) were immobilized by physical adsorption
method. The effects of various immobilization and drying process parameters on the
residual activity and the protein loading of the immobilized enzyme preparation were
studied and thereby different optimum preparations were observed for different
enzymes. Under the optimum immobilization conditions the residual activity achieved
with YADH, LBADH, and ADH T was about 80 %, 316 %, and 325 %, respectively.
The hypothesis of bubble nucleation as a cause for loss of enzyme activity during the
low pressure drying process was verified. The effects of various gas-phase reaction
conditions on the initial reaction rate and the half-life of the optimized preparations
were also studied. It was observed that addition of a suitable buffer (50 mM phosphate
buffer, pH 7) or an optimum amount of sucrose (5 times greater than the amount of
protein on weight basis) during the enzyme immobilization enhanced the half-life of the
immobilized enzymes in the gas-phase reaction. Water activity significantly influenced
the initial reaction rate and the half-life of the immobilized enzyme preparations in the
gas-phase reaction. The optimum water activity found for LBADH and ADH T was the
same (0.55). Under the optimized immobilization and gas-phase reaction conditions the
thermo-stability of the ADH enzymes was enhanced tremendously. The space-time
-1 -1⋅ ⋅yield of (R)-phenylethanol was about 1000 gm l d with LBADH and the space-time
-1 -1S)-phenylethanol was about 600 gm ⋅ l ⋅ d with ADH T. The total turn over
5 5number of LBADH was about 91× 0 and the same of ADH T was about 31× 0.






DEDICATED TO MY FAMILY




ACKNOWLEDGEMENTS
I experience great pleasure to convey my profound sense of gratitude and respect to
Prof. Dr.-Ing. Jochen Büchs, the chair of Biochemical Engineering of RWTH-Aachen,
for his excellent supervision, encouraging discussions, and inspiring suggestions.
I wish to extend my appreciation to Dr.-Ing. Antje Spiess and Dipl.-Ing. Torsten
Natelberg of our institute and Dr.rer.nat. Nagrao Rao of Rane Rao Reshamia
Laboratories Private Limited (India) for their keen interest and fruitful suggestions on
this research work.
I wish to thank the technical staffs of our institute for their cooperation in this research
work, Institute of Biotechnology of RWTH-Aachen for giving me permission to use the
sonicator and vacuum pump. I also wish to thank to Institute of Ceramic Components of
RWTH-Aachen for analyzing a few samples using the scanning electron microscopy
and supplying a few supports for enzyme immobilization.
I also thank my students Daniela, Simone, and Tobias for their help in carrying out
some experiments in this research work.
Special thanks go to my colleagues and friends for their kind help and ready assistance.
The financial support from Deutsche Forschung Gemeinschaft (Graduiertenkollegs 440
‘‘Methods in Asymmetric Synthesis’’) for the period from October 2001 to September
2004 is highly acknowledged.
Every wheel needs an axis to keep on rotating. In my case, my axis was and it is my
parents – source of my all inspirations. They shouldered the burdens of every little
problem just to make me successful in this work. There were also my brothers, sister-in
laws, Abhishek, divyanshu, and my friends Manisha and Dipayan who played very
significant roles on different aspects of my life through out my stay in abroad. Any
attempt to thank them would defy the greatness of their contributions.

Archana H. TrivediTABLE OF CONTENTS
Glossary of Abbreviations and Symbols ix
List of Tables xi
List of Figures xii
Preface xvi

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 5
2.1 Alcohol dehydrogenase enzymes 6
2.1.1 Yeast alcohol dehydrogenase 7
2.1.2 Lactobacillus brevis alcohol dehydrogenase 8
2.1.3 Thermoanaerobacter species alcohol dehydrogenase 8
2.2 Definition of immobilized enzymes 8
2.3 Advantages and disadvantages of enzyme immobilization 8
2.4 Classification of enzyme immobilization methods 9
2.5 Optimization of enzyme immobilization parameters 9
2.6 Principle of enzymatic gas-phase reaction 11
2.7 Characteristics of enzymatic gas-phase reaction 12

3 MATERIALS, METHODS, AND EXPERIMENTAL SET-UP 17
3.1 Materials 18
3.1.1 Enzymes, cofactors, supports, and other chemicals 18
3.2 Methods 19
3.2.1 Preparation of cell extract 19
3.2.2 Pre-treatment of supports 19
3.2.3 Immobilization of enzyme by physical adsorption 20
3.2.4 Determination of protein 21
3.2.5 Determination of enzyme activity 22
3.2.6 Determination of enzyme stability 24
3.2.6.1 Aqueous phase 24 Table of Contents
3.2.6.2 Gas-phase 24
3.2.7 Scanning electron microscopy 25
3.2.8 Reaction system 26
3.2.9 Batch gas-phase reaction 26
3.2.10 Continuous gas-phase reaction 27
3.2.11 Gas chromatography 28
3.2.11.1 Preparation of liquid samples 28
3.2.11.2 Preparation of gas samples 28
3.2.11.3 Off-line analysis 28
3.2.11.4 On-line analysis 29
3.3 Experimental set-up 30
3.3.1 Batch gas-phase reactor 30
3.3.2 Continuous gas-phase reactor 37

4 RESULTS AND DISCUSSION 39
4.1 Optimization of enzyme immobilization parameters 40
4.1.1 Effect of temperatures on immobilization efficiency 40
4.1.2 Effect of stirring speed and stirring time on
immobilization efficiency 41
4.1.3 Effect of drying conditions on
immobilization efficiency 42
4.1.4 Effect of additives on immobilization efficiency 48
4.1.4.1 Sucrose and others 48
4.1.4.2 Buffers 52
4.1.5 Effect of amount of added protein on
immobilization efficiency 56
4.1.6 Effect of supports on immobilization efficiency 58
4.2 Evaluation of batch gas-phase reactors 62
4.2.1 Selection of a batch reactor 62
4.2.2 Functionality of the selected batch reactor 65
4.3 Optimization of continuous enzymatic gas-phase
reaction parameters 67
4.3.1 Effect of total gas flow rate on initial substrate conversion and
initial reaction rate of immobilized enzyme preparation 67
viiTable of Contents
4.3.2 Effect of water activity on initial reaction rate and half-life
of immobilized enzyme preparation 68
4.3.3 Effect of additives on initial reaction rate and
half-life of immobilized enzyme preparation 70
4.3.3.1 Buffers 70
4.3.3.2 Sucrose and others 77
4.3.4 Effect of amount of added protein on initial reaction rate
of immobilized enzyme preparation 81
4.3.5 Effect of cofactor-to-protein molar ratio on initial reaction
rate and half-life of immobilized enzyme preparation 83
4.3.6 Effect of temperature 86
4.3.6.1 Initial reactio