Вы находитесь на странице: 1из 3

Communications

Use of Bioanalytical Systems for the ion-exclusion and ion-exchange chromatography [2,3]. Using
Improvement of Industrial Tryptophan the enzyme tryptophan synthetase as biocatalyst and pyridox-
al phosphate as co-enzyme the conversion of L-serine and
Production* indole to L-tryptophan is performed. In this patented process
a wild-type mutant of Escherichia coli, which constitutively
By Roland Ulber, Carsten Protsch, Dörte Solle, overexpress the tryptophan synthetase [4,5], is used. The
Bernd Hitzmann, Birgit Willke, Robert Faurie, and biocatalyst is suspended in the L-serine solution and the indole
Thomas Scheper** is added in a fed-batch mode. For this process a reliable on-line
process control is absolutely necessary according to the
following facts:
1 Introduction ± High indole concentration inhibits the enzyme reaction and
± nonoptimal concentration levels influence side reactions
Amino acids are used in different areas. The most important
which decrease the product quality and quantity or require
applications can be found in food industry (50 %), animal feed
additional down-stream procedures.
(30 %) and as pharmaceutical ingredients (20 %). Amino
The concentration level of L-serine, indole and tryptophan
acids are used in food industry because most of them enhance
in the biotransformation process is monitored via an on-line
the taste and flavor of food products. For different animals
HPLC system with an analysis frequence of three analyses per
(e. g. pigs and poultry) fifty percent of the amino acids are
hour. However, a fast and reliable analysis method is
essential and in most animal feed products (e. g. soy, fish meal,
necessary to detect not only the substrate and product
or grain) these essential amino acids, like L-methionine,
concentration. The overall reaction kinetics, the activity of
L-lysine and L-threonine, are missing. Due to this fact these
the biocatalyst (enzyme activity) should be detected addi-
amino acids are added to the feed products. In pharmaceutical
tionally within the complete production process. For this
products amino acids of high purity are necessary. In this area
purpose a biosensor system was developed to monitor the
they are used as pre- and post-surgery nutrition. A standard
chromatographic separation of serine from sugar beet
infusion consists of eight amino acids which are essential for
molasses, which is the first step of the L-tryptophan
human beings (L-methionine, l-phenylalanine, L-threonine,
production [2,3,6]. For the second step, the biotransformation
L-tryptophan, L-lysine, L-isoleucine, L-leucine and L-valine)
of L-serine and indole, a 2-D fluorescence spectrometer is
and some nonessential amino acids (e. g. glycine, L-glutamic
adapted to the process. This fluorescence spectrometer can
acid, L-serine, L-proline and L-alanine). L-tryptophan is used
detect all fluorophores in the reaction solution within one
as a drug according to its sedative qualities as antidepressant
minute. Based on chemometric models, the concentration of
and neuroleptic. In the cosmetic industry high-quality skin
the substrates and the product can be predicted based on the
creams are also based on amino acids.
fluorescence data as well as metabolic information (enzyme
For the production of amino acids several industrial
activity). Using this combination of model and measurement,
methods are used:
an optimal control of the indole feeding profile is possible.
± Extraction from renewable resources
± Fermentation±Chemical synthesis
± Biotransformation
About 3 billion tons of amino acids are produced worldwide 2 Method and Materials
by these methods [1]. The company AMINO GmbH,
Frellstedt, Germany, uses renewable resources, such as sugar The biotransformation process from indole and serine to
beet molasses, for the production of amino acids for tryptophan is performed in several steps at the AMINO
pharmaceutical applications. These amino acids are gained GmbH. The final bioreactor has a volume of 10,000 liters.
by chromatographic separation and biotransformation reac- Using a bypass, the on-line HPLC system and the 2-D
tions. For the biotechnological production of L-tryptophan, fluorescence spectrometer (BioView, Delta Light & Optics)
L-serine and indole are used as substrates. The L-serine is are coupled to the process. The spectrometer is interfaced to
isolated by AMINO GmbH from sugar beet molasses using the process via liquid fibre cables. In this way it can be used
± also in explosion-protected production areas. The principal
[*] Lecture given at the ACHEMA 2000 in Frankfurt, Germany. setup of this device is given in Fig. 1. The 2-D spectro-
[**] Dr. R. Ulber. Dipl.-Chem. C. Protsch, Dipl.-Chem. D. Solle, PD Dr. B. fluorometer uses independent filter wheels for excitation and
Hitzmann, Prof. Dr. T. Scheper, Institut für Technische Chemie, emission up to 16 different filters each which can be designed
Universität Hannover, Callinstraûe 3, D-30167 Hannover; B. Wilke, R.
Faurie, AMINO GmbH, An der Zucker-Raffinerie 10, D-38373 Frell- individually. Usually, measurements in steps of 20 nm are
stedt, Germany. performed (excitation range: 270±550 nm, emission range:

Eng. Life Sci. 1 (2001) 1, Ó WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2001 0930-7516/01/0107-0015
1618-0240/01/0107-0015$$17.50+.50/0
17.50+.50/0 15
Communications

be identified, which are important for the analysis of


tryptophan, indole and serine. Fig. 3 shows the result of such
a sensitivity analysis. Black areas indicate high and white areas
indicate low sensitivity for the given analyte. Using the
subtraction spectra and the sensitivity analysis two fluores-
cence areas can be determined and can be correlated to the
increase of the fluorescence activity in the area of the indole/
tryptophan region and to the decrease of the fluorescence in
the area of the serine fraction (fluorescent byproducts).
Figure 1. Schematic set-up of the 2-D fluorescence spectrometer (Delta Light With this information the concentration profile of trypto-
& Optics, Denmark). phan, indole and serine during the biotransformation process
can be predicted with a high precision using a chemometric
310±590 nm), which was found most suitable without any loss model [8]. In Figs. 4 and 5 the concentration profiles of these
of information. At the beginning of the monitoring of a analytes are given in comparison to off-line HPLC analysis.
complete 2-D fluorescence spectrum, the excitation filter The off-line samples were taken every 5 minutes at the start of
wheel is set to the first filter. The excitation light is guided via the process and then every 20 minutes. The chemometric
the fiber optic into the measuring chamber of the bypass. The model uses the off-line and the fluorescence data (analysis
backward fluorescent light is monitored via the emission filter frequence of 1 minute). The correlation between the predicted
which switches from filter to filter for a whole rotation cycle. and measured data is in the range of 5 %. Only at the end of the
Afterward, the excitation filter wheel
switches to the next position and the
emission spectrum of the fluorescence
produced by this defined excitation
wavelength is monitored by the next
complete cycle of the emission filter
wheel. This procedure is repeated
until the excitation filter wheel has
performed one complete cycle. After-
ward, a new spectrum can be mea-
sured. A complete spectrum can be
collected within 60 seconds. In addi-
tion, it is possible to monitor single
wavelength pairs continuously.

3 Results and Discussion

Subtraction spectra, as shown in Fig.


2, are used to detect changes within the
biotransformation. These spectra are Figure 2. 2-D Fluorescence subtraction spectra of the biotransformation from inole and serine to tryptophan
(end of process ± start of process); black areas in the subtraction spectra indicate decreasing and white areas
obtained by the substraction of each 2- increasing fluorescence activity.
D fluorecence spectrum minus the
spectrum of the start of the process.
Black areas in the subtraction spectra
indicate decreasing and white areas
increasing fluorescence activity dur-
ing the biotransformation procedure.
Indole and tryptophan are fluoro-
phores present in various concentra-
tions in the reactor. In addition, some
components (byproducts) of the ser-
ine fraction used have high fluores-
cence intensities. Serine itself does not
fluoresce.
Using sensitivity analysis special
areas in the substraction spectra can Figure 3. Sensitivity analysis for (A) tryptophan and (B) indole (white: low sensitivity, black: high sensitivity).

16 Ó WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2001 1618-0240/01/0107-0016 $ 17.50+.50/0 Eng. Life Sci. 1 (2001) 1
Communications

process the difference between the predicted and measured 4 Acknowledgements


tryptophan concentration increases (about 15±20 %) (Figs. 4
and 5). Financial support of this research provided by Deutsche
Bundesstiftung Umwelt (Osnabrück, Germany, Az. 13028/01)
is gratefully acknowledged.
Received: January 22, 2001 [K 2820]

References
[1] Leuchtenberger, W., Amino Acids ± Technical Production and Use, in:
Biotechnology (H. J. Rehm, G. Reed, Eds.), VCH, # 1996, pp. 455±502.
[2] Sosnitza, P.; Irtel, F.; Ulber, R.; Busse, M.; Faurie, R.; Fischer, L.;
Figure 4. Concentration profile for tryptophan and serine during the Scheper, T., Biosensors for Supervision of Industrial Chromatographic
biotransformation procedure (measured and predicted data). Downstream Processing in Biotechnology, Biosensors Bioelectron. 13
(1998) pp. 1251±1255.
[3] Ulber, R.; Faurie, R.; Sosnitza, P.; Fischer, L.; Stärk, E.; Harbeck, C.;
Scheper, T., Monitoring and Control of Industrial Downstream
Processing of Sugar Beet Molasses, J. of Chromatography A 882
(2000) pp. 329±334.
[4] Wagner, F.; Klein, J., Verfahren zur mikrobiellen Herstellung von
L-Tryptophan, DE 2 841 642, 1980.
[5] Faurie, R.; Fries, G., From Sugar Beet to Lyphan, in: Basic Aspects and
Practical Applications, Advances in Experimental Medicine and Biology
(T. Simat, Ed.) Plenum Press, New York 1999.
[6] Irtel, F.; Sosnitza, P.; Busse, M.; Faurie, R.; Fischer, L.; Ulber, R.;
Scheper, T., Langzeiteinsatz von Biosensorsystemen zur Überwachung
industrieller Chromatographieprozesse, Chem.-Ing.- Tech 72 (2000)
pp. 502±505.
[7] Marose, S.; Lindemann, C.; Scheper, T., Biotechnol. Prog. 14 (1998)
Figure 5. Concentration profile for indole during the biotransformation pp. 63±74.
procedure (measured and predicted data). [8] Hitzmann, B.; Pekeler, T.; Lindemann, C.; Marose, S.; Scheper, T.,
Chemometric Models for the On-Line Estimation of Bioprocess
Variables from 2-D Fluorescence Spectra, in: Computer Applications in
Biotechnology: a Proceedings Volume from the 7th IFAC Int. Conf.
Using the 2-D fluorescence spectroscopy a fast and reliable (T. Yoshida, S. Shioya, Eds.) Osaka Japan, May 31/June 4, 1998,
Pergamon Press, Kidlington 1998.
analytical method is available for the on-line monitoring of
industrial process.
This paper was also published in German in Chem. Ing. Tech. 73 (2001) No. 5,
At present, the model can only be used for the monitoring pp. 524±526.
and prediction within one production cycle. In future, a better _______________________
model should be used which enables predictions of the
concentration from one biotransformation process to the
following biotransformation processes. Using such a model a Optimization of Biocatalysts
reliable control system for the conversion of indole and serine
for Technical Processes
to tryptophan is planed. The following process optimization is
possible:
± A higher product yield leads to a reduction of the substrates By Martina Pohl*
serine and indole.
± A higher product concentration leads to better conditions
for the following downstream process (less product specific 1 Problem
energy).
± In addition, the amount of wastewater can be decreased. The increasing interest in biocatalysts (enzymes) for the
Using the described systems (2-D fluorescence sensor and production of (chiral) fine chemicals is based on their ability to
chemometric model) already a 20 % higher product concen- catalyze reactions with high selectivity at room temperature
tration can be achieved. This leads in regard to the following and in aqueous media. This allows the reduction in established
downstream process to a reduction of 200 to 300 of steam chemical synthetic routes and avoids undesired by-products.
(20 % of the product-specific energy costs). Using the To meet these requirements, a high catalytic activity combined
optimized process conditions up to 2000 m3 wastewater can with high selectivity and, most importantly, a good process
be avoided (corresponding to 0.4 tons COD) per year. stability are required.
This project demonstrated that the use of innovative
bioanalytical systems, such as the 2-D fluorescence spectros- ±
copy, leads to better economical and ecological conditions of [*] Dr. M. Pohl, MPB Cologne GmbH, Neurather Ring 1, D-51063 Köln,
an industrial biotransformation process. Germany.

Eng. Life Sci. 1 (2001) 1, Ó WILEY-VCH Verlag GmbH, D-69469 Weinheim, 2001 0930-7516/01/0107-0017
1618-0240/01/0107-0017 $$ 17.50+.50/0
17.50+.50/0 17