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Industrial Production of Amino Acids by Microorganism and Fermentation!
About 20 amino acids are synthesized in the cell of the most microorganisms. They are utilized in the synthesis of proteins and other essential substances required by the cell. Fermentative production of amino acids has started by the discovery of glutamic acid producing bacterium, Corynebacterium glutamicum (Micrococcus glutamicum), by Kinoshita et al. (1957).
Since then much research work has been carried out on fermentative production of amino acids. Large number of microorganisms were isolated from nature which are capable of producing amino acids by fermentation in commercially feasible quantities, especially from the auxotrophic bacteria (table 5.1).
The details of production of different amino acids by fermentation involving different microorganisms are precised in table 5.2.
The general layout of fermentation plant for production of amino acids is illustrated in Fig. 5.1.
Of the various amino acids, L-glutamic acid and L-lysine are in great demand for commercial production. Auxotrophs are used in the fermentation production of these two amino acids. The major portion of the amino acid accumulation occurs after log phase of growth of the microorganism. The concentration of specific growth factors is very crucial for the maximum yield of the product because concentration above or below the optimum substantially, reduces the yield.
Microorganisms, generally, do not produce amino acids in surplus or more than required quantities. They accomplish this by regulating cellular metabolism. However, overproduction of the amino acids by microorganisms can be achieved by controlling the complex regulatory systems. This is done by raising auxotrophs by mutation.
The enzymes responsible for the regulatory effect or repressor of an amino acid are made inactive due to mutation which leads to the accumulation of the particular amino acid in commercially feasible quantities which is being exploited commercially for fermentation (table 5.2).
Apart from fermentative processes, some amino acids can be synthesized quite economically by chemical processes. However, these chemical processes generally yield, D-isomers of amino acids, which are biologically inactive and cannot be used as food flavouring substances or food supplements. Only L-isomers are useful as food supplements or flavouring substances and, therefore, most of the L-isomer of amino acids are produced by fermentation process. Some of the amino acids produced by microbial fermentation and their annual production are precised in table 5.3.
1. Extraction of protein hydrolysates;
2. Direct fermentation;
3. Microbial transformation of precursors; and
4. Use of enzymes or immobilized cells.
Economic Importance:
Aspartame (L-aspartyl-L-phenylalanine methyl ester) which is made up of phenylalanine and L-aspartic acid is used as low-calorie sweetener in soft drinks. Many amino acids are used in medicine particularly as ingredients of infusion solutions in post-operative treatments. In chemical industry, amino acids are used as starting materials for the manufacture of polymers such as polyalanine fibers and lysine-isocyanate resins.
Polymethylglutamate is used as a surface layer in the manufacture of synthetic leather. N-acetyl derivatives of some amino acids are used in the manufacture of cosmetics and as surface active substances. Urocanic acid is used as a sun-tanning agent and is produced by the transformation of histidine. Glycine is used in the manufacture of an herbicide, glycophosphate.
L-lysine, 2, 6-diaminohexanoic acid, is synthesized by microorganisms either via diaminopimelic acid pathway or the aminoadipic acid pathway. However, in any single organism only one of the two alternative pathways is employed. Bacteria, actinomycetes, cyanobacteria (Blue-green algae), some phycomycetes and protozoa use the DAP (Diaminopimelic acid) pathway (Fig. 5.2), while some phycomycetes, all ascomycetes and basidiomycetes and eukaryotic algae uses the aminoadipic acid pathway.
L-lysine occurs in plant proteins only but too low in concentration. Addition of L-lysine can, therefore, increases the quality of food. The market for L-lysine is increasing day by day. Though, L-lysine is produced today only by microbial processes, a variety of approaches for its production have been developed.
L-glutamic acid is a dicarboxylic amino acid and contains two carboxyl groups, along with an amino-group which is attached to the α-carbon atom. Fermentative production of L-glutamic acid was started after the isolation of Corynebacterium glutamicum in 1957. Before that, it was used to be produced by chemical synthesis and the product used to contain a mixture of D and L- glutamic acid.
Subsequently L-glutamic acid production was found to occur in a wide variety of bacteria like Corynebacterium, Brevibacterium, Microbacterium and Arthrobacter. Streptomycetes, yeasts and fungi are also reported to be capable of producing L-glutamic acid upto 30 g per liter.
Corynebacterium glutamicum which produces good amount of L-glutamic acid possesses the following characteristics:
1. The bacterium is gram (+) positive, non-sporulating and non-motile.
2. Requires biotin for growth.
3. Shows little activity of α-ketoglutaric acid dehydrogenase.
4. Shows increased activity of glutamate dehydrogenase.
Mutants of C. glutamicum secrete L-glutamic acid in large quantities even in the presence of high concentration of biotin. For example – a lysozyme sensitive mutant of C. glutamicum is able to convert 40% of the added carbon source to L-glutamic acid even in the presence of 100 mg per liter of biotin.
L-aspartic acid is widely used as a food additive and in pharmaceuticals. Since the time of its use in Aspartame as an artificial sweetener production, its demand increased considerably. Although L-aspartic acid was originally produced exclusively using aspartase due to high
productivity and cost effectiveness of the process. In fact, this method proved to be highest enzyme used in biotechnology. This method allows the production of 2,20,000 kg of L-aspartic acid per kg of enzyme. The reaction is inter-convertible (Fig. 5.10).
In fact reaction favours the ammonification. The enzyme of E.coli is a tetramer with a molecular weight of 1,96,000 daltons and has absolute requirement of divalent cation. However, the enzyme was unstable in the beginning which could be overcome by immobilization in polyacrylamide. Subsequently carrageenan, which has half-life for about two years proved to be better material for immobilization of aspartase.
The initial disadvantage of this enzyme was that it converts part of fumaric acid to L-malic acid. Heat treatment of cells eliminated fumarase activity almost completely using such conditioned and starting with 1m ammonium fumarate. Using such conditioned cells it was possible to produce 987 mM L-aspartic acid 10.7 mM non- reacted fumarate and only trace quantities of L-malic acid of 1.9 mM.
For production of L-aspartic acid, the immobilized cells of E.coli are packed into a column designed as a multi stage system. The stages introduced consisting of horizontal tubes serve two purposes. On one hand, they allow effective cooling to prevent decay of the catalytic activity since the asparatase reaction is exergonic. On the other hand, the flow proportionately of the column are increased. Any compacting of bed over time is prevented and the preferred plug flow characteristics are obtained.
The continuous process enables full automation and control to achieve the optimum output with the highest product quality. Yet another advantage of such controlled continuous process reduces waste production. It is estimated that about 3.4 tonnes of aspartic acid per day in a column of 1000 d-1 which is 100 tonnes per month. The final product is eventually purified by crystallization.
L-Phenylalanine can be produced with E. coli or C. glutamicum. The pathway of L-phyenylalanine synthesis is shared in part with that of L-lyrosine and L-tryptophan. These three aromatic amino acids have in common. The condensation of erythrose – 4 – phosphate and phosphoenol pyruvate to deoxyarabinoheptulosonate phosphate (DAHP) with further conversion in six steps to choristmate. L-Phenylalanine is then finally made in three further steps (Fig. 5.11).
There are three DAHP synthase enzymes in E.coli encoded by aroF, aroG and aroH. These enzymes play key role in flux control and regulation of catalytic activity in each case by one of the aromatic amino acids. About 80% of the total DAHP synthesis activity is contributed by aroG-encoded enzyme. The increased flux towards L-phenylalanine can be obtained by over expression of either aroF or aroG encoding feed-back resistant enzymes.
Further, more phe A over expression is essential which encodes the bifunctional chorismate mutase pre phenate dehydratase. A second chorismate activity is present as a bifunctional chorismate mutase – prephenate dehydrogenase. The pre A-encoded enzyme activities are inhibited by L-phenylalanine and pre A expression is dependent on the level of t-RNA. A pre phenylalanine producer obtain as per rule are tyrosine auxotrophic mutants.
Fermentation:
As with other amino acids effective phenylalanine production is the joint result of engineering the cellular metabolism and control of production process. Control is necessary for two reasons. First, the carbon flux has to be optimally distributed between the four major products of glucose conversion which are phenylalanine, biomass, acetic acid CO2.
The second reason is that cellular physiology is not constant during the course of E.coli and tends to produce acetic acid which has strong negative effect on the process efficiency, which can be prevented by sugar feeding, regulating O2 concentration, sugar consumption and biomass concentration. Glucose should be added when it is totally exhausted at stage-2 of fermentation. Thus, feeding rate is a compromise where the process run at highest possible feeding rate.
When the tyrosine initially present has consumed, the cells proceeds to stage 3. At this stage, the metabolic capacity of the cells decreases which brings about a consequent decrease of glucose feeding rate. At the end of stage 3, acetic acid excretion begins and the cells enter stage 4 where no further phenylalanine accumulation occurs and the process eventually get terminated (Fig. 5.12).
Thus, it reveals that sophisticated feeding strategy with adaptive control stimulates a very high phenylalanine concentration and can be achieved with a high yield of phenylalanine per liter alongwith a yield of 27.5% carbon.