Biotechnological production of podophyllotoxin by linum album suspension cultures [Elektronische Ressource] / by Hermann Jörn Garden

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2003
• Nombre de lectures: 97
• Langue: English
• Poids de l'ouvrage: 153 Mo

Extrait

BIOTECHNOLOGICAL
PRODUCTION OF PODOPHYLLOTOXIN
BY Linum album SUSPENSION CULTURES






Inaugural-Dissertation
submitted to the Faculty of Natural Sciences
Heinrich-Heine-University Düsseldorf
in fulfilment of the requirements for the degree of
Dr. rer. nat.










by
Hermann Jörn Garden
from Kamp-Lintfort

Düsseldorf
2003






































Printed with permission of the Faculty of Natural Sciences, Heinrich-Heine-
University Düsseldorf, Germany. This thesis was prepared with financial
support of the „Graduiertenförderung NRW“, as well as the EU-project:
BIO4-CT98-0451 – Lignocancer.



Referee: Prof. Dr. A.W. Alfermann
Co-Referee: Prof. Dr. P. Westhoff
Oral Examination: 15.12.2003




CONTENTS


A. Introduction 1
A.1 General aspects of natural products and their application 1
A.2 Plant-derived antineoplastic agents – the case of podophyllotoxin 2
A.3 Occurrence and function of secondary metabolites 6
A.4 Lignans and lignin as products of the phenylpropanoid metabolism 8
A.5 Application of plant cell culture in pharmaceutical biotechnology 11
A.5.1 Historical development 11
A.5.2 Problems associated with large-scale plant in vitro culture 12
A.5.3 Potential of plant cell culture for the production of secondary metabolites 13
B. Scope of the thesis 17
C. Material and Methods 19
C.1 Plant material and culture conditions 19
C.2 The bioreactor system 19
C.2.1 Design of the airlift bioreactor 20
C.2.2 Setup and operation of the bioreactor system 21
C.3 Analytical procedures 23
C.3.1 Characterization of the oxygen-transfer capacity - k a determination 23 L
C.3.2 Measurement and quantification of culture-growth 25
+ - 3-C.3.3 Analytical procedures for NH , NO and PO quantification 26 4 3 4
C.3.4 Atomic-absorption spectroscopy 27
C.3.5 Extraction and quantification of podophyllotoxin 27
C.3.6 Crude protein extraction and measurement of enzyme activity 28
D. Results & Comments 29
D.1 Preliminary studies 29
D.1.1 Cell growth and production dynamics of Linum album cell suspensions 29
D.1.2 Initial obstacles of the bioreactor culture 31
D.1.3 Determination of the oxygen transfer rates 34
D.2 Effect of the oxygen supply on podophyllotoxin production – the shake flask system 35
D.2.1 Monitoring the culture oxygen saturation during the growth period 35
D.2.2 Influence of different gyratory shaker speeds on podophyllotoxin production 36
D.2.3 Physiological responses to different culture volumes 38
D.3 Effects of the oxygen supply on podophyllotoxin production – the bioreactor system 45
D.3.1 Influence of the aeration rate on podophyllotoxin accumulation 45
D.3.2 Studies on the bioreactor culture at an elevated oxygen saturation 52
D.4 Influence of the inoculum on enzyme activity and ptox accumulation 57
D.4.1 Effects of different inoculum densities on shake flask culture 57
D.4.2 Effects of the inoculum density on bioreactor culture 59

I D.5 Optimisation of the culture medium 63
D.5.1 Adjustment of the culture medium concentration 63
D.5.2 Effects of fed-batch culture and high oxygen saturation 69
D.5.3 Influence of macronutrients on enzyme activity and podophyllotoxin accumulation 73
D.5.4 Improved podophyllotoxin production in a revised culture medium 77
D.6 Selection of a high-producing cell line 82
D.6.1 Preliminary shake flask studies on different cell lines 83
D.6.2 Characterization of a high-producing cell line in the bioreactor system 85
D.7 Summery of results 88
E. Discussion 93
E.1 Basic studies on culture characteristics - the shake flask system 93
E.1.1 Dynamics of biomass formation during the growth period 93
E.1.2 Podophyllotoxin accumulation by Linum album (X4) cell suspensions 95
E.2 Annotations on culture stress and medium discolouration 96
E.3 Dependency of the podophyllotoxin accumulation on oxygen supply 98
E.3.1 Increase of the shaker speed 99
E.3.2 Variation of the culture volume inside the shake flasks 99
E.3.3 Impact of the aeration rate 101
E.3.4 Enhanced podophyllotoxin production by supplementation of pure oxygen 102
E.4 Adjustment of the inoculum density 104
E.5 Implications of the culture medium on podophyllotoxin production 106
E.5.1 Effects of an increased nutrient supply – dynamics of podophyllotoxin accumulation 106
E.5.2 Effeeay – phenylpropanoid metabolism activity 108
E.5.3 Modification of the inorganic nitrogen composition 109
E.5.4 Comparison of the Linum album system with Podophyllum cultures 111
E.6 Selection of a high-producing cell line 112
F. Summary 113
G. List of Instruments & Components 117
H. Bibliography 121














II


LIST OF ABBREVIATIONS

ATP adenosine triphosphate
BR bioreactor
∆C oxygen-concentration gradient
CAD cinnamyl alcohol dehydrogenase
4CL 4-coumarate:CoA ligase
CoA coenzyme A
DO dissolved oxygen saturation
dw dry weight
ε extinction coefficient
EC Enzyme Commission
FDA fluorescein diacetate
fw fresh weight
G growth index
kDa kilodalton
k a volumetric oxygen transfer coefficient L
L. Linnaeus, Carolus
mM millimol
mS millisiemens
MS Murashige & Skoog
NADP nicotinamide adenine dinucleotide phosphate
OTR oxygen transfer rate
PAL phenylalanine ammonia lyase
L-Phe L-phenylalanine
Pi inorganic phosphate
ptox podophyllotoxin
rpm rotations per minute
t mass doubling time d
L-Trp L-tryptophan
L-Tyr-tyrosine
vvm aeration volume per culture volume
∆x boundary layer
µ specific growth rate
Y growth yield x/s







III
A. INTRODUCTION
A.1 General aspects of natural products and their application
Throughout the ages, humans have been exploiting plants for the production of foodstuff, clothing,
flavours, fragrances and, last not least, medicines. For the treatment of human ailments elaborate
healthcare systems evolved in countries like China, India and the tropical areas around the world.
Up to now plant-derived drugs and remedies for primary health care have played an important role
particularly in the rural parts of developing countries. It has been estimated by the World Health
Organization (WHO) that approximately 80% of the world’s population depends on traditional
medicine and even half of the most-prescribed drugs in the US is linked directly to natural product
research (NEWMAN, et al. 2000). During the last centuries highly sophisticated methods have been
developed to find out the effective principle of a single phytopharmacon, known for its beneficial
potential, or proved in vitro activity. In fact, many isolated natural products such as digoxin,
morphine, reserpine and quinine have replaced the source from which they were derived. One of the
most prominent examples is the discovery of salicylic acid in Filipendula ulmaria L. and in the bark
of Salix alba L. – plants whose pain-relieving properties were described hundreds of years ago. As
prolonged usage of salicylic acid causes stomach problems further chemical modification was
thneeded to obtain aspirin in the middle of the 19 century (MANN & PLUMMER 1993). Apart from
this simplistic view that a single constituent accounts for the pharmacological function of a drug, in
many cases investigations failed to depict the active principle of one single plant extract. In this
context PHILLIPSON (1999) stated that the antimalarial herb Artemisia annua L. contains many other
compounds in addition to artemisinin which in vitro enhances the activity against Plasmodium
falciparum. Another well-known example is ginseng (Panax ginseng C.A.Mey.), which has been
used in traditional Chinese medicine for over 2000 years. Most of the pharmacological benefits of
ginseng (stimulatory effects on the central nervous system, immunmodulatory properties) could
have been attributed to the steroid-like group of ginsenosides. However, overall effects can be quite
complex due to the variable composition of structurally diverse ginsenosides, numerous intrinsic
modes of action and the occurrence of non-steroid constituents (ATTELE, et al. 1999).
Despite the fact that plant based complementary medicine has recently gained wide public
recognition it has to be pointed out that natural products often contain toxic compounds and
unpredictable side effects. Pregnant women, children and elderly patients, in particular, are mainly
at risk because of false body weight dose estimation and because of the poor hepatic enzyme
activity for biotransformation (CHAN 1994; CHANG, et al. 2000). Moreover phytochemists are
concerned about inadequate quality control, lack of prove of efficiency and the non-critical attitude <