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
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1.1 Essential Amino Acids: L-phenylalanine
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2.0 Production of L-phenylalanine by Enzymatic Method
2
3.0 Upstream Processing
4
4.0 Downstream Processing
5
5.0 Conclusion
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6.0 References
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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 1
(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 α-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
Ammonia
acid Aerobic Fermentation of
Immobilization
PAL-bearing cells
of cells
Bioreactor
Recycle
Recovery
Purification
L-phenylalanine
Figure 3: Block Flow Diagram of the Process (Humg-Yu et al., 1988; Swann, 1985)
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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: 15-25 oC; 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 0°C to about 30°C at least through the last portion of the conversion process. Preferably the temperature is from about 5°C to about 25°C. 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, adjusti ng 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
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