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LITERATURE REVIEW
glutamic acid extracellular. Among many isolates we found a colony that might
be fit for the purpose, named this isolate Micrococcus glutamicus No.534.
that biotin must play a key role in the physiology of the cells and their
stages, we found that the cell form can change considerably. For this reason,
with discoveries regarding key regulatory features, it was found that many
[Ikeda.K, 2002].
Amino acids produced by such a process are all in their natural (L) form
and this gives microbial production a big advantage over chemical synthesis.
Thus, a new industry called amino acid fermentation was born. The
constituent amino acids. The new process, on the contrary, was a biosynthetic
process using carbohydrate and ammonium ions. Therefore our process can
contribute to the amino acid supply and also helps to increase the absolute
increase year by year, so will the demand for amino acids and protein. After
World War II, two new fermentation industries were born in Japan. These are
back to the year 1908. At that time Prof. Kikunae Ikeda at the University of
Tokyo found that monosodium glutamate (MSG) had a potent taste enhancing
During these studies he found a small crystal. This was glutamic acid, which
solution and tasted again. Surprisingly, it had changed into a beautiful taste.
That was the aim of his studies, since he was searching for the potent essence
various foods, their taste was noticeably improved [Kinoshita et.al., 1987]. His
real intention was to improve nutrition and increase the short life expectancy of
the Japanese at that time. He finally got the idea that even if the same food was
eaten, its value might be increased if the taste is enhanced. In this sense, an
professor Ikeda began to search for the essence of good taste. Konbu had been
traditionally used in Japanese food as a taste enhancer, so he believed it
should contain the essence of flavor. This led to the discovery of MSG, whose
properties for the daily food of the Japanese [Kinoshita et.al., 1987].
Mr.Saburosuke Suzuki was the man who supported Prof. Ikeda’s desire.
Wheat gluten was chosen as the raw material to obtain MSG. But this task was
very difficult. Concentrated HCl must be used for decomposition of gluten, but
no anticorrosive vessels were available in those days. So clay pots were used,
but they were fragile and their use was very dangerous. Moreover, the gas from
HCl caused serious damage to the health of the residents living near the
produce MSG continued for ten years, before he finally became confident of
commercial success. Once MSG appeared in the market, its miracle power
overwhelmed the food market and it became an essential food additive. Mr.
Suzuki’s company is now known as Ajinomoto Co., Inc [Ikeda .K, 1908].
After World War II, Dr. Benzaburo Kato set up Kyowa Hakko Co., Inc. in
1945. Because of the shortage of food, the Japanese suffered great hunger.
Everywhere malnourished patients were seen. Dr. Kato was deeply worried by
this situation and thought of an idea for relieving the miserable situation by
fermentation process.
Amino acids are the building blocks that constitute proteins. Which are
very important to living systems for their survival Amino acids comprise about
16% N2 that distinguish them carbohydrates and fats in the body
[Lichtenberger, 1996]. Amino acids are the building blocks used to make
proteins and peptides .With the exploitation of new uses and the growing
markets of amino acids, production of amino acid technology has made large
progress during the latter half of the 20th century. Fermentation technology
has played a crucial role in the progress made and currently the fermented
value. This area is highly competitive in the world market and process
As the building blocks of life, amino acids have played an important role
in both human and animal nutrition and health maintenance [Balch et.al.,
molecules, although of course lipids and carbohydrates are also essential for
life. On account of its functionality and the special features arising from
great interest for the chemical industry [Leuchtenberger, 1996]. Of the twenty
standard protein amino acids, the nine are essential the rest of the amino acids
competition between these two fields to produce amino acids in a cheap and
energy reducing mode. Amino acids have many special properties which make
them very valuable, as for example their contribution to nutrition, the taste,
proteinogenic amino acids are the building blocks of proteins, they are
important intermediates on the pathway from the genetic to the protein level.
1995. Divided in 38% for food, 54% for feed and 8% for other applications
R C COOH
NH2
but for many of them it would be much more profitable to produce with
different methods. The advantage of the enzymatic synthesis and the direct
histidine and L-cysteine for example are produced by extraction from protein
immobilized cells.
The barrier for multi enzyme systems is reached when the effectiveness
side reactions and by-products. On this account the direct fermentation is the
preferable process in commercial aspects for L-lysine and L-Glutamic acid [Kole
seed proteins, especially the prolamines, yielded 20-45 percent of glutamic acid
on hydrolysis. The man who first noted in 1908 the commercial importance of
could convert non proteinaceous raw material into an amino acid and excrete it
the medium. This left no doubt that glutamic acid produced was the result of
metabolism. At that time, a two step L-Glutamic acid production process was
used. The glutamic acid producing bacterium first discovered was reported as
form species, all potent strains, were subsequently isolated by many others.
more of the glutamic acid secreting bacteria can be easily found in nature as
cultures were used for glutamic acid production due to increasing demand for
medium.
and release the product in the extracellular environment. The isolated soil
surround the entire cell as a structured layer [Eggling et.al., 2001]. The wild
type strains are mostly able to grow aerobically on basic minimal media
addition biotin due to the fact that this bacterial species is completely biotin
sporulating. Since the isolation in 1957 high amounts of L-Glutamic acid have
been produced with new developed or advanced strains of this species [Eggling
et.al., 2001].
structure which is different from other gram positive bacteria the peptidoglycan
mycolic acid layer is linked again with the arabinogalactan [Eggling et.al,
2003].
about this organism and its metabolic fluxes in context of amino acid
bacterial strain for amino acid overproduction. It has been observed that the
regulatory system is much simpler than that of Escherichia coli [Tosaka et.al.,
et.al., 1961], L-lysine [Eggling et.al., 1999] and L-valine [Blombach et.al., 2007]
factors for the cultivation process in order to reach high amounts of L-Glutamic
acid are the optimal concentration of biotin to influence and support cell
[Clement et.al., 1986]. Another important factor to prevent side reactions and
1. Phosphoenolpyruvate carboxylase.
2. Pyruvate kinase.
3. Pyruvate carboxylase.
4. Pyruvate dehydrogenase.
5. Citrate synthetase.
6. Aconitase.
7. Isocitrate dehydrogenase.
8. L-glutamate dehydrogenase (GDH).
9. α-ketoglutarate dehydrogenase (KDH).
10. Isocitrate lyase.
11. Malate synthethase.
Fig. 2.4: Regulation of L-Glutamic acid biosynthesis in
of the enzymes GDH and KDH (Figure 2.4). In overproducers the conversion
than the side reaction of the substrate with KDH which leads back to the citric
acid cycle [Shiio et.al, 1980]. In Figure 2.4 the versatile regulation mechanisms
in biological pathways for L-Glutamic acid (feedback inhibition and repression)
directions are quite obvious due to the complex city and various connections in
Apart from sugars, amino acids are common substrates for microbial
cell growth. Sources of amino acids, like peptones, tryptones and casamino
acids are used in laboratory media. The degradation of individual amino acids
depends on the availability of other energy sources and on the C/N ratio.
The first step in biochemical amino acid degradation is, in general, the
removal of the α-amino acid group. The reaction products are 2-oxo acids.
There are different biochemical ways to perform this reaction. They are, as
follows:
- The transamination
- The β-elimination
After the α-amino group removal further degradation steps take place
according to the back bones of the 2-oxo acids (aliphatic, heterocyclic and
aromatic compounds).
which are important in this thesis is shown in Figure 2.4. The amino acids can
bodies. Lysine, leucine and threonine belong to this group of amino acids
which can be degraded only this way. The degradation products of these three
leucine and phenylalanine are amino acids which can be metabolized on both
biochemical ways for degradation, the ketogenic and the glucogenic one. The
other amino acid groups, shown in Figure 2.4, are degraded through the
glucogenic ways. This means that their degradation products are available for
largely imported amino acid in India and the total worldwide production of L-
Glutamic acid is more than 1.5 million tons per year through fermentation.
being pursued to improve the glutamic acid production especially under the
always been an issue which the scientific community have pursued with zeal.
Glutamic acid. In the year 1866, L-Glutamic acid was discovered by Karl
Heinrich Leopold Ritthausen. Later in the year 1907, Kikunae Ikeda identified
many foods especially in seaweed and he termed this flavor Umami [Ikeda,
like wheat proteins, soya bean proteins upon acid hydrolysis with HCl was first
In the same year Donald A. Kita and Jackson Heights N.Y produced L-
In 1959, Kwei-chao chao and J.W Foster reported that, the Bacillus
Glutamic acid.
L-Glutamic acid can be produced by the fermentation of saccharide
member of this group desthio biotin, biotin disulfoxide and biocytin and the
yield exceeds 4.0 g/dl.This was first reported by shinichi motozaki et.al. in
1963.
glutamicus M-560 was developed by Thomas Philips in 1963. The yield was 40
g/L when grown in 3L molasses medium containing 37.5 parts per billion of
14.3 g/L and Corynebacterium acetoacidophilum ATCC No. 13870 produced 7.3
yield of L-glutamic acid was 6.3 g/L in the G-medium and 2.1 g/L, 1.9 g/L,
4.0 g/L and 2.8 g/L in the H-medium containing these hydrocarbons.
John D.Douros, Jr., West Chester et.al. in 1965, described a method for
Utilizing hydrocarbons under aerobic conditions at 300c for 48-96 hrs. The
yield obtained was 1-4 g/L for n-decane with in 36 hrs of incubation time.
L-Glutamic acid was produced from unsaturated fatty acids which were
reported by Hisoyoshi Okazaki et.al. in the year 1967. The organisms used
were Bacillus thiogenitalis No. 653 and its Oleic acid requiring mutant D-248.
mg/ml of L-Glutamic acid but at the same biotin concentration glutamic acid
production by No.563 was reduced to zero. It was also found that No.563
the medium.
maximum amount of L-Glutamic acid i.e more than 50% (w/w) of initial sugar
content present in the medium containing wheat bran extract or rice bran
ATCC 14067.
Haruo Momose and Takashi Takagi in 1978 reported that glutamic acid
produced glutamic acid after a temperature shift from 300C to 370C.One typical
mutant strain, TS-88 produced 2 g/dl of glutamic acid in a biotin rich beet
with ethanol at increased concentrations from 5 g/L to 25 g/L during fed batch
culture and the yield of L-Glutamic acid reached to 26 g/L. The organism used
was Brevibacterium divaricatum NRRL 2311. This method was first explained
glutamic acid. When these resistant strains grown in 10 g/dl of glucose the
yield obtained was in between 5-41% and it was 5-33% when resistant strains
grown in g/dl of cane molasses. But at varied temperatures 31.50C, 350C, 370C
in presence of 3.6 g/dl of cane molasses the yield of glutamic acid was in the
medium. The same strains produced 47-50 g/L of glutamic acid when grown in
ATCC 13032 which gave the highest yield of 13.7 g/L. The culture medium also
16-18 g/L of glutamic acid. These strains have liberated 49-51 g/L of glutamic
acid in case molasses at 31.50C for 36 hrs of incubation time [Yoshimura et.al,
1985].
It was reported that, an immobilized Corynebacterium glutamicum grows
used for the production of L-Glutamic acid. The conversion of glutamic acid
The maximum yield of glutamic acid 6.86 mg/ml was obtained when 2%
glucose medium was fermented for 48 hrs by a Brevibacterium Sp. was reported
[ Nampoothiri et.al1995].
carbon source and produced 88 g/L of glutamic acid [ Das K et.al, 1995].
Glutamic acid 3.0 g/L, 5.0 g/L, 18.5 g/L and 20.0 g/L respectively [N.Tujimoto
et.al, 1995].
glucose medium.
enriched with 10% glucose, urea, mineral salts and vitamins for the production
during shift up temperatures 340C, 370C, 390C and the yield was 0.0, 0.5, 0.9
g/dL for ATCC 13869 and it was 5.8, 8.3, 9.2 g/dL for the strain AJ13029 by
utilizing glucose as carbon source. But during time intervals 8hrs, 12hrs,
16hrs produced 0.5, 0.1, 0.0 g/dL of glutamic acid by ATCC 13869 and 8.3,
sorbitan monopalmitate etc yielded 56% of L-glutamic acid and the productivity
liberated 56% of L-glutamic acid with a productivity of 6.6g/L in 40hrs and the
was sufficient and 70% of glutamic acid was liberated. In glucose medium
shift up from 330C to 370C, 380C, 390C, 400C and 410C causes the
et.al. 2004].
The investigations carried out by Jyothi et.al. in the year 2005, revealed
The experimental study carried out by yugandhar N.M et.al. During 2007
revealed that the maximum yield of L-Glutamic acid 40.5 mg/ml was obtained
with Brevibacterium roseum free cells under optimum parameters. The glutamic
acid at a concentration of 37.2 mg/ml and 39.6 mg/ml were obtained with
into carrageenan gel beads. The best yield was obtained 75.7% at a
S. Y. Pasha et.al in the year 2011, Comparative studies were carried out
on glutamic acid production with wild type cells, mutants, immobilized cells
and immobilized mutants of Corynebacerium glutamicum. Immobilization was
physical mutants and five chemical mutants were selected for study.
Fermentation was carried out for a period of six days at 300C at 200 rpm.
mutants. The maximum yield was wild type 14 g/l, physical mutant at 32.6 g/l
The study of Mahmud Tavakkoli et.al. in the year 2012 concluded the
fact that date waste juice was the best substrate for the production of glutamic
acid. They used Corynebacterium glutamicum CECT 690 culture and response
Glutamic acid production. The maximum yield was 39.32 mg/ml which was
determined by this model and in the second stage the yield was 118.75 mg/ml,
142.25 mg/ml and 95.83 mg/ml at three different air flow rates.
mixture for the commercial and pilot scale production of L-Glutamic acid,
nitrates, peptones and other amino acids may also be utilized. [Nishida et.al.,
1979].
inorganic salts of various metals like magnesium sodium, potassium and also
[Delauney s.et.al.,1999].
2.9 L-Glutamic acid fermentation technology
aspartic acid and L-alanine are among other amino acid produced
products. The growing market for amino acid produced with Corynebacteria led
as in molecular biology. During the last decade big efforts were made to
increase the productivity and to decrease the production costs [Westrin I.A,
1990].
paved the way for the success of the fermentation technique in amino acid
production. It was advantageous here that the wild strain could be used on an
strains have been investigated in depth [Minoru Yoshimura et.al., 1982]. The
source, such as sugar cane syrup, as well as the required nitrogen, sulfur, and
MSG (1.5 million tons) is currently produced each year by this method, making
L-Glutamic acid the number one amino acid in terms of production capacity
been used for industrial production of amino acids. Market development has
been particularly dynamic for the flavor-enhancer glutamate and the animal
feed amino acids L-lysine, L-threonine, and L-tryptophan, which are produced
glutamicum and Escherichia coli from sugar sources such as molasses, sucrose,
or glucose. But the market for amino acids in synthesis is also becoming
increasingly important, with annual growth rates of 5–7%. The use of enzymes
also of suitable plants, to arrive at even more efficient processes for amino acid
nonessential amino acid that the body uses to build proteins. It is also the
system when aminated, glutamic acid forms the important amino acid
glutamine. Because it has a carboxylic acid moiety on the side chain, glutamic
acid is one of only two amino acids (the other being aspartic acid) that have a
net negative charge at physiological pH. This negative charge makes glutamic
acid a very polar molecule and it is usually found on the outside of proteins
a rate of 6.2 percent compared to the other amino acids. Glutamic acid is also
system. Glutamic acid helps transport potassium into the spinal fluid and is
proteins yield as much as 45% of their weight as glutamic acid. Glutamic Acid
acid are soy, meat, poultry, fish, eggs, and dairy products. When glutamic acid
1. Dairy products
2. Meat
3. Poultry
4. Fish
is used by the body to build proteins. It can attach itself to nitrogen atoms in
the process of forming glutamine, and this action also detoxifies the body of
across the blood brain barrier, although it does not pass this barrier that
Parkinson's, and mental retardation. The fluid produced by the prostate gland
contains significant amounts of glutamic acid, and this amino acid may play a
role in normal function of the prostate. Glutamic acid may have protective
glutamate (MSG), the form of glutamic acid that is used as a flavor enhancer,
effects (including headache, fatigue, and depression) [Reeds P.J. et.al, 2000].
transportation of potassium across the blood brain barrier, although itself does
not pass this barrier that easily. It also shows promise in the future treatment
fuel in the brain, and can attach itself to nitrogen atoms in the process of
forming glutamine, and this action also detoxifies the body of ammonia. This
action is the only way in which the brain can be detoxified from ammonia. The
fluid produced by the prostate gland also contains amounts of glutamic acid,
and may play a role in the normal function of the prostate [ Delauney .S et.al.,
2002].
metabolism.
Dosage based on the results from clinical studies with positive results,
daily dosage of Glutamic Acid ranges from between 2 - 15 grams (high doses
may produce symptoms like Headache and Neurological problems) required
for lipid and glucose metabolism [Reeds P.J. et.al. ,2000]. Therapeutic Uses
are as follows
With the advent of new uses and the growing markets of amino acids,
amino acid production technology has made significant progress during the
latter half of the 20th century. Amino acid industry has been expanding and
further reduce the production costs, thereby increasing the world rapid
low cost fermentation processes for many kinds of amino acids and the recent
the amino acid industry. Fermentation technology has played crucial roles
over a period of time and currently the amino acid produced by fermentation
compared to the process using free cells in batch mode. The entrapment of
cells in sodium or calcium alginate gel beads is very useful procedure because
simplicity of the method, low price and non toxicity [Devlin T.M, 2002].
Due to high demand of L-Glutamic acid all over the world, for
most research work confined to free cell fermentation gap existing between
free cell batch reactor studies and immobilized cell reactor studies. Since
significant research has not been carried out in immobilized cell reactor there
is a vast scope to carry out research work for producing L-Glutamic acid by