CBE667 Industrial Bioprocess Technology

INDIVIDUAL ASSIGNMENT

Industrial Production of L-phenylalanine by Enzymatic Method

Prepared by
Mohd Shahrizi Razali
2012401476
EH2426B

Prepared for
Mrs. Suhaila Mohd Sauid
Lecturer for CBE667
Faculty of Chemical Engineering
UiTM, Shah Alam

21 April 2014

CONTENTS

Body of Report

Page

1.0 Introduction

1.1 Essential Amino Acids: L-phenylalanine

2.0 Production of L-phenylalanine by Enzymatic Method

3.0 Upstream Processing

4.0 Downstream Processing

5.0 Conclusion

6.0 References

1.0 INTRODUCTION
According to Ivanov et al. (2013), amino acids play an important role in human nutrition and
health maintenance. Nowadays amino acids are used as animal feed additives, flavour
enhancers, ingredients in cosmetic and pharmaceutical products and as specialty nutrients
in the medical field, and the production capacity requirements are constantly increasing. Van
Balken (1997) added that amino acids are versatile chiral (optically active) building blocks
for a whole range of fine chemicals. Moreover, concern with regard to the exposure of man
and his environment to an ever increasing number of chemicals. These led to the arising of
usage and demand for therapeutic agents, pesticides, food and feed additives that are
exhibit less toxic side-effects and are more environmentally acceptable. Amino acids can be
produced by protein hydrolysis, chemical synthesis or biotechnological methods (Ivanov et
al., 2013). To this end a central role will be played by chiral compounds, as nature at the
molecular level is intrinsically chiral. Consequently, this provides an important stimulus for
companies to market chiral products as pure optical isomers. This in turn results in an
increasing need for efficient methods for the industrial synthesis of optically active
compounds (van Balken, 1997). Biotechnology methods for the industrial production of
amino acids are of three types: use of microbial enzymes or immobilized cells (enzymatic
method), semi-fermentation, and direct fermentation (Ivanov et al., 2013). Biotechnology
methods especially enzymatic route offers advantages like produces optically pure D- and Lamino acids at high conversion with less by-products and no racemization occurs during
synthesis (Ikeda, 2003).
1.1

Essential Amino Acids: L-phenylalanine

Based on Ehrlich (2013), phenylalanine is an essential amino acid (a building block for
proteins in the body), meaning the body needs it for health but cannot make it. It is
obtained from food. Phenylalanine is found in 3 forms: L-phenylalanine (L-phe), the
natural form found in proteins; D-phenylalanine (a mirror image of L-phe that is made
in a laboratory), and DL-phenylalanine, a combination of the two forms.
Along with the same line, according to article titled Analysis of L-phenylalanine
in China (2012), L-phe helps the brain to produce important chemicals called
neurotransmitters. These chemicals keep the brain functioning correctly in many ways.
Figure 1 shows the chemical structure of L-phe. It is found in most foods that contain
protein such as beef, poultry, pork, fish, milk, yogurt, eggs, cheese, soy products
(including soy protein isolate, soybean flour, and tofu), and certain nuts and seeds
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(Ehrlich, 2013). The main uses for L-phe includes for synthesis of aspartame (primary
raw materials), manufacturing of amino acids for feed and food additives,
pharmaceutical intermediates, synthetic vitamins and supplements and so on. Besides
these, it is also used in the synthesis of anticancer drugs, antivirals, vitamins B6 and
so on (Analysis of L-phenylalanine in China, 2012). Noted that L-phe mainly for
aspartame making, the reason for this is that both D- and L-phenylalanine are
enantiomers of sucrose and both equally sweet, but only naturally occurring Denantiomers is metabolised in the body; making the synthetic L-enantiomer a dietary
sweetener (van Balken, 1997).

Figure 1: Chemical Structure of L-phenylalanine (van Balken, 1997)

2.0 PRODUCTION OF L-PHENYLALANINE BY ENZYMATIC METHOD

The current interest in the production of L-phe as precursor for aspartame production has
led to the development of numerous competing biotransformation reactions utilizing different
types of enzymes and substrates. Humg-Yu et al. (1988) had listed enzymatic routes to Lphe as tabulated in Table 1.
Table 1: L-phenylalanine production by enzymatic synthesis (Humg-Yu et al., 1988)
Transamination
Phenylalanine dehydrogenase/formate dehydrogenase
Phenylalanine dehydrogenase/hydroxyisocaproate dehydrogenase
Phenylalanine dehydrogenase/high pressure hydrogen
-Acetamidocinnamic acylase/phenylalanine dehydrogenase/lactate dehydrogenase
Phenylalanine ammonia lyase
Hydantoinase
For the sake of this report, only the industrial production of L-phe from trans-Cinnamic
Acid by phenylalanine ammonia lyase enzyme will be discussed. The principle of the
process which is the reaction scheme is shown in Figure 2. According to Swann (1985),
enzymatic methods using L-phenylalanine ammonia-lyase for the conversion of trans2

cinnamic acid to Lc-phe generally comprise of several steps: firstly (1) aerobically
propagating a phenylalanine ammonia lyase (PAL)-producing microorganism in an aqueous
nutrient medium until substantial amounts of PAL are produced, secondly (2) contacting the
cells of the PAL-producing microorganism from step (1), either as the whole culture broth or
separated cells there- from, or the isolated enzyme, with ammonium ions and transcinnamate ions and allowing the reaction to proceed under controlled temperature and pH
conditions until the conversion to L-phenylalanine approaches equilibrium and thirdly (3)
separating and recovering the L-phenylalanine from the reaction mixture. Block flow diagram
(BFD) presenting the flow of the process that includes upstream and downstream
processing is shown in Figure 3. This enzymatic method was used by GENEX
CORPORATION in United States of America to produce several hundred tons per year of Lphe during 1984 and 1985 (Humg-Yu et al., 1988).

Figure 2: Enzymatic Reaction of the Process (van Balken, 1997)

Trans-cinnamic
acid
Aerobic Fermentation of
PAL-bearing cells

Immobilization
of cells

Ammonia

Bioreactor

Recycle

Recovery

Purification

L-phenylalanine

Figure 3: Block Flow Diagram of the Process (Humg-Yu et al., 1988; Swann, 1985)

3.0 UPSTREAM PROCESSING

Based on Figure 3, the upstream processing includes fermentation, immobilization and
biotransformation in the bioreactor. Under this upstream section, first process is the
production of propagation of microorganism bearing the PAL enzyme. The PAL can be
obtained from various overproducing strains of yeast Rhodoturula for example like strains R.
glutinis, R. rubra, or R. graminis (Fotheringham, 1999; Naito et al., 1991). The fermentation
should be conducted in aerobic environment in the growth-promoting condition.
Conventional method should be employed for growing the cells. Cells are inoculated into a
nutritional medium containing sources of carbon and nitrogen and essential vitamins,
minerals and other growth factors that can be utilized by the desired cells (Swann, 1985).
Complex media can be used for this purpose. Swann (1985) also stated that after the cell
reached desired cell density, they are induced to make PAL under PAL-inducing conditions
(at temperature: 15-25oC; pH: 5.5-7.5). PAL induction is generally achieved by adding small
amounts of a compound that acts as a substrate for the PAL like L-Phenylalanine itself, D,Lphenylalanine, L-tyrosine, and D,L-tyrosine. This inducing step can prolong until preferably
PAL activity reached 2.0 units/mL (Swann, 1985). In this process, fermented cells of yeast
strains R. glutinis, R. rubra, or R. graminis were recovered and washed before bioreaction
under batch or immobilized conditions by known procedures on a solid support that can be
reused for so long as the enzyme activity is maintained (Fotheringham, 1999).
The second step which is the immobilization of cells can be carried out accordingly to
Nelson (1976). According to the patent holder, microbial cells can be effectively immobilized
by chemical covalent bonding of the cells to water-insoluble particulate polymer matrix. The
chemical bond can be formed either with the preformed polymer or with reactive monomer
prior to polymerization. Further, treatment of the cells with a polyfunctional cross-linking
agent either prior to, during or after bonding reduces enzyme loss from the cell (Nelson,
1979). Cells can be immobilized in polyacrylamine, K-carrageenan gel or vermiculite, to
mention a few.
Thirdly the biotransformation of the enzymatic conversion of trans-cinnamic acid and
ammonia to L-phe is preferably conducted in a plug flow reactor since immobilized cells can
be packed into. By this way, the reaction can ran continuously over the immobilized cells on
a solid support. This reaction should be is maintained at a temperature of from about 0C to
about 30C at least through the last portion of the conversion process. Preferably the
temperature is from about 5C to about 25C. Typically, the reaction is run at the higher
temperature until conversion approaches about 70% (Swann, 1985).
4

4.0 DOWNSTREAM PROCESSING

Based on Figure 3, the downstream processing covers all process after enzymatic reaction
in bioreactor. According to Fotheringham (1999), L-phe is typically recovered only from the
extracellular medium and washed cells. Because lysis of cells to recover additional
phenylalanine is not generally practical, L-phe remaining within the cells after washing is
usually lost. Since cell biomass is extremely high in large-scale fermentations, this can
represent up to 5% of total phenylalanine produced. This is also supported by Swann (1985)
as the author stated that L-phe can be recovered from the reaction mixture by any suitable
means. For example, solids can be removed by filtration or centrifugation to produce a
clarified solution, and L-phe can be precipitated from that solution by adjusting the pH to the
isoelectric point of L-phe to about 5.5, for example.
Parallel to that, Naito et al. (1991) had outlined that the isolation of L-phe from the
reaction mixture obtained in a reaction using cells having PAL activity can be carried out, for
example, by a process which comprises the following steps;
(1) Centrifuging the reaction mixture to remove the cells
(2) Heating the resultant solution to eliminate any excess ammonia
(3) Adding an acid to the solution to provide an acidic pH and centrifuging or filtering
the thus pH- adjusted solution, whereby any remaining cinnamic acid precipitate is
removed
(4) Causing the resultant solution to flow through an ion-exchange resin to adsorb Lphe, followed by eluting L-phe
(5) Concentrating the resultant eluate containing L-phe, adjusting the pH of the residue
to the isoelectric point (5.5) of L-phe, and then collecting precipitated L-phe by a
separating process including filtration or the like.
5.0 CONCLUSION
All in all, it can be concluded that there are numerous commercial processes have been
developed for the large-scale commercial production of L-phe since the early 1980s, fueled
by the enormous increase in L-phe demand for the dipeptide sweetener, aspartame.
Enzymatic method in production of L-phe indeed offers advantages over other production
means like produces pure product at relatively high efficiency with less by-products.
However, the development of numerous competing biotransformation reactions has yet to
replace the direct fermentation method. Thus a way forward strategy surely needed for this
method to overcome the challenges so for it to be widely used in industry.
5

6.0 REFERENCES
Analysis of L-phenylalanine in China. (2012). Retrieved April 19, 2014, from
http://www.fjmd.com.cn/index.php?/detail/mdnewsen/41?lang=en
Ehrlich,
S.
D.
(2013).
Phenylalanine.
Retrieved
April
19,
2014,
from
http://umm.edu/health/medical/altmed/supplement/phenylalanine
Fotheringham, I. G. (1999). Synthesis of Phenylalanine by Fermentation and
Chemoenzymatic Methods. In Ager, D. J. (Eds.). Handbook of Chiral Chemicals. Marcel
Dekker Inc.: NY
Humg-Yu, H., Walter, J. F., Anderson, D. M. and Hamilton, B. K. (1988). Enzymatic
Production of Amino Acids. In Biotechnology and Genetic Engineering Reviews. Vol. 6.
Intercept Ltd.: Dorset, UK
Ikeda, M. (2003). Amino Acid Production Processes. In Scheper, T, Faurie, R. and
Thommel, J. (Eds.). Advances in Biochemical Engineering and Biotechnology. Vol. 79.
Germany: Springer
Ivanov, K., Stoimenova, A., Obreshkova, D. and Saso, L. (2013). Biotechnology In The
Production Of Pharmaceutical Industry Ingredients: Amino Acids. Biotechnology and
Biotechnological Equipment. 27 (2): 3620 3626
Naito, N., Koito, M., Ura, D., Fukuhara, N. (1991). Production Process for L-phenylalanine.
European Patent Application No. 91106275.0
Nelson, R. P. (1976). Immobilized Microbial Cells. U. S. Patent No. 3957580
Swann, W. E. (1985). Production of L-phenylalanine. European Patent Application No.
85304128.3
Van Balken, J. A. M. (1997). Biotechnological Innovations in Chemical Synthesis. Oxford:
Reed Educational and Professional Publishing