Industrial production of chemical acids: glutamine acid

Background

Proteins are large, complex molecules that are critical for the normal functioning of the human

body. They are essential for the structure, function, and regulation of the body’s tissues and organs.

Proteins are made up of hundreds of smaller units called amino acids that are attached to one

another by peptide bonds, forming a long chain. You can think of a protein as a string of beads

where each bead is an amino acid. Amino acids are biologically important organic compounds

containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side-chain (R

group) specific to each amino acid. The key elements of an amino acid are carbon, hydrogen,

oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids.

About 500 amino acids are known (though only 20 appear in the genetic code) and can be classified

in many ways. They can be classified according to the core structural functional groups' locations

as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino acids; other categories relate to polarity,

pH level, and side-chain group type (aliphatic, acyclic, aromatic, containing hydroxyl or sulfur,

etc.). In the form of proteins, amino acids comprise the second-largest component (water is the

largest) of human muscles, cells and other tissues. Outside proteins, amino acids perform critical

roles in processes such as neurotransmitter transport and biosynthesis.

In biochemistry, amino acids having both the amine and the carboxylic acid groups attached to

the first (alpha-) carbon atom have particular importance. They are known as alpha or α-amino

acids (generic formula H2NCHRCOOH in most cases, where R is an organic substituent known

as a "side-chain"); often the term "amino acid" is used to refer specifically to these. They include

the protein-genic ("protein-building") amino acids, which combine into peptide chains

polypeptides to form the building-blocks of a vast array of proteins. These are all L-stereoisomers

("left-handed" isomers), although a few D-amino acids ("right-handed") occur in bacterial

envelopes, as a neuromodulator (D-serine), and in some antibiotics. Twenty of the proteinogenic

amino acids are encoded directly by triplet codons in the genetic code and are known as "standard"

amino acids. The other three "non-standard" or "non-canonical" are selenocysteine (present in

many noneukaryotes as well as most eukaryotes, but not coded directly by DNA), pyrrolysine

(found only in some archea and one bacterium) and N-formylmethionine (which is often the initial

amino acid of proteins in bacteria, mitochondria, and chloroplasts). Pyrrolysine and selenocysteine

are encoded via variant codons; ex, seleno-cysteine is encoded by stop codon and SECIS element.

Codon–tRNA combinations not found in nature can also be used to "expand" the genetic code and

create novel proteins known as alloproteins incorporating non-proteinogenic amino acids.

The amino acid business is a multi-billion dollar enterprise. All twenty amino acids are sold,

albeiteach in greatly different quantities (Table 1). Amino acids are used as animal feed additives

(lysine,methionine, threonine), flavor enhancers (monosodium glutamic, serine, aspartic acid) and

asspecialty nutrients in the medical field. Glutamic acid, lysine and methionine account for the

majority, by weight, of amino acids sold. Glutamic acid and lysine are made by fermentation;

methionine is made by chemical synthesis. The major producers of amino acids are based in Japan,

the US, South Korea, China and Europe. Many microbe-based industries have their origins in

traditions that go back hundreds or thousands of years. The amino acid industry has its roots in

food preparation practices in Japan. Seaweeds had been used for centuries there and in other Asian

countries as a flavoring ingredient. In1908, Kikunae Ikeda of Tokyo Imperial University isolated

the flavor enhancing principle from the seaweed konbu (also spelled kombu, Laminaria japonica;

related to kelp) as crystals of monosodium glutamate (MSG). Adding MSG to meat, vegetables

and just about any other type of prepared food makes it savory, a property referred to as umami.

Soon after Ikeda’s discovery, and recognizing the market potential of MSG, Ajinomoto Co. in

Japan began extracting MSG from acid-hydrolyzed wheat gluten or defatted soybean and selling

it as a flavor enhancer.

Production of MSG via “fermentation” grew out of the ashes of WWII in Japan. Around 1957,

Japanese researchers led by S. Kinoshita at Kyowa Hakko Kogyo Co. isolated soil bacteria that

produced large amounts of glutamic acid. Producing strains were found by inoculating soil isolates

in a grid pattern on duplicate Petri plates. The colonies were allowed to grow and one set of

duplicates was killed with UV irradiation. The killed plate was overlaid with soft agar containing

a Leuconostoc mesenteroides. Since L. mesenteroides required glutamic acid for growth, it only

grew in the vicinity of colonies that had excreted glutamate. Potential glutamate producers were

then picked from the duplicate, unkilled, plate. Members of the Actinobacteria in the genus

Corynebacterium (originally named Micrococcus glutamicus) were the most effective producers.

Over the years, various glutamate-producing bacteria have been isolated and classified as

Arthrobacter, Brevibacterium, or as members of other genera, but recent work has shown that

almost all of these strains belong to the genus Corynebacterium. Wild-type cultures produced up

to 10 g/l glutamic acid. Yields were quickly improved by process engineering and by developing

over-producing mutants. Yields are now in excess of 100 g per liter. The isolation of bacterial

glutamate-producers led to the development of large-scale manufacture of MSG from cheap sugar

and ammonia rather than from more expensive extracts of plants or animals. In the early 1960s,

workers at the same company found that C. glutamicum homoserine auxotrophs (see below)

produced lysine thus providing the first viable fermentation process for lysine production. Today

these bacteria produce well over 1,000,000 metric tons of MSG and 600,000 metric tons of lysine

annually.

Importance of Amino acids

- Active Pharmaceutical Ingredients (APIs), there are pharmaceutical products that make use of

amino acids themselves, and products that are synthesized and manufactured using amino acids

as starting materials. Amino Acids are also used in infusion solutions, in addition to tablet and

granulated forms, and are an essential part of modern medical treatments.

- Medical Foods / Medical Nutrition, amino Acids are used as a way to provide a concentrated,

specific and efficient intake of required nutrient components in medical foods for malnourished

patients, elderly people with lower digestive capabilities, as well as in other uses.

- Dietary Supplement / Health Foods / Functional Foods & Beverages, amino Acids are used for

compensating amino acid deficiencies, as well as in supplements that make use of the specific

function of amino acids. Products are sold in tablet, granular and capsule forms. These products

were once mostly used by athletes, but they are now popular among the wider public for

everyday workout and health maintenance.There are also functional food and beverage

products that contain amino acids for functions similar to those found in health foods.

- Cosmetics, amino Acids are used in cosmetics for their unique moisturizing effect and pH

levels. Amino acid derivatives are also used for their stability and absorbency.

- Culture Medium, recently, pharmaceutical products research and manufacture using cell

culture process have become very popular. Amino Acids are essential components of cell

culture medium. Amino Acids are considered to be indispensable to cell growth and production

of antibodies and proteins.

Glutamic acid

Glutamic acid, also referred to as glutamate (the salt of glutamic acid), is an acidic, α-amino

acid that is found in many proteins, and that in free form functions as an important

neurotransmitter, a metabolic intermediate in the Krebs cycle, and a compound that can

combine with an help in the elimination of toxic ammonia from the body. The salt monosodium

glutamate (MSG) is a common food additive and flavor enhancer. Along with aspartic acid, to

which is behaves similarly, glutamic acid is classified as an acidic amino acid. The L-isomer,

which is the only form that is involved in protein synthesis, is one of the 20 standard amino

acids common in animal proteins and required for normal functioning in humans. However, it

is not considered to be an "essential" amino acid since it does not have to be taken in with the

diet, but can be synthesized by the human body from other compounds through chemical

reactions. Glutamic acid is responsible for one of the human senses of taste, termed umami,

adding to the classical taste sensations of sweet, salty, sour, and bitter. Umami applies to the

sensation of savoriness, the detection of glutamates in such foods as meats, cheese, and other

protein-heavy foods. Beyond its practical value, taste adds to the human enjoyment of creation,

joining such diverse senses as being able to see various colors, hear different sounds, smell a

vast array of odors, and so forth. Such senses allow interaction with nature and touch upon the

inner aspect of people. As a source for umami, MSG, the sodium salt of glutamic acid, is used

to enhance the flavor of foods. Glutamic acid's three letter code is Glu, its one letter code is E,

and its systematic name is 2-Aminopentanedioic acid (IUPAC-IUB 1983). A three-letter

designation for either glutamic acid (Glu) or the amino acid glutamine (Gln) is Glx—this is

often used in cases in which peptide sequencing reactions may convert glutamine to glutamate

(or vice versa), leaving the original identity of the amino acid in doubt.

Chemical Structure

In biochemistry, the term amino acid is frequently used to refer specifically to alpha amino

acids: those amino acids in which the amino and carboxylate groups are attached to the same

carbon, the so-called α–carbon (alpha carbon). The general structure of these amino acids is:

Most amino acids occur in two possible optical isomers, called D and L. The L amino acids

represent the vast majority of amino acids found in proteins. They are called proteinogenic

amino acids. As the name "proteinogenic" (literally, protein building) suggests, these amino

acid are encoded by the standard genetic code and participate in the process of protein

synthesis. In glutamic acid, only the L-stereoisomer is involved in protein synthesis in

mammals. Glutamic acid's chemical formula is HOOC-CH(NH2)-(CH2)2-COOH (very

similar to aspartic acid's formula, HOOC-CH(NH2)-CH2-COOH), but with an extra CH2), or

more generally C5H9NO4. (Aspartic acid's general forumula is C4H7NO4.) Glutamic acid

behaves similar to aspartic acid, but has a longer, slightly more flexible side chain. As its name

indicates, it is acidic, with a carboxylic acid component to its side chain. Generally either the

amino group will be protonated or one or both of the carboxylic groups will be deprotonated.

At neutral pH all three groups are ionized and the species has a charge of -1. The pKa value

for Glutamic acid is 4.1. This means that at pH below this value it will be protonated (COOH)

and at pH above this value it will be deprotonated (COO-).

Application of glutamic acid

Food Production

- L-Glutamic Acid is widely used as nutritional supplement in food production.

- As flavor enhancer: in MSG and spices to improve flavor.

- As nutritional supplement: in food industries to provide amino acids.

Beverage

- L-Glutamic Acid is widely used as flavor enhancer in beverage.

- As flavor enhancer: in soft drink and wine to improve flavor.

Pharmaceutical

- Not enough is known about application of L-Glutamic Acid in Pharmaceutical.

Cosmetics

- L-Glutamic Acid is widely used as Hair restorer in Cosmetics.

- As Hair restorer: in treatment of Hair Loss.

- As Wrinkle: in preventing aging.

Agriculture/Animal Feed

- L-Glutamic Acid is widely used as nutritional supplement in Agriculture/Animal Feed.

- As nutritional supplement: in feed additive to enhance nutrition.

Other Industries

- L-Glutamic Acid is widely used as intermediate in various other industries.

- As intermediate: in manufacturing of various organic chemicals.

Biosynthesis of Glutamic acid

An amino acid precursor is converted to the target amino acid using 1 or 2 enzymes, allows the

conversion to a specific amino acid without microbial growth, thus eliminating the long process

from glucose. Raw materials for the enzymatic step are supplied by chemical synthesis the enzyme

itself is either in isolated or whole cell form which is prepared by microbial fermentation, as

following:-

Reactants Products Enzymes

Glutamine + H2O → Glu + NH3 GLS, GLS2

NAcGlu + H2O → Glu + Acetate (unknown)

α-ketoglutarate + NADPH + NH4+ → Glu + NADP+ + H2O GLUD1, GLUD2

α-ketoglutarate + α-amino acid → Glu + α-oxo acid transaminase

1-pyrroline-5-carboxylate + NAD+ + H2O → Glu + NADH ALDH4A1

N-formimino-L-glutamate + FH4 ⇌ Glu + 5-formimino-FH4 FTCD

Industrial Production and use of Microorganisms

L-amino acids are major biological components commercially used as additives in food, feed

supplements, infusion compounds, thera-peutic agents and precursors for peptides synthesis or

agriculture based chemicals. The amino acids are the second most important category, after

antibiotics, with fermentation products exhibiting the highest growth rates. L-glutamic acid was

the first amino acid produced commercially. The substance was discovered and identified in the

year 1866 by the German chemist Karl Heinrich Leopold Ritt-hausen. L-glutamic acid was mainly

produced by microbial fermentations and the chemical mode of synthesis is not widely preferred

due to the formation of racemic mixture.

Corynebacterium glutamicum is a very important fermentative bacteria most widely know for

its role in the production of monosodium glutamate, or MSG. Discovered in 1957 in Japan as a

natural producer of glutamic acid, C. glutamicum is a Gram positive, facultatively anaerobic,

heterotrophic bacterium with an irregular rod shape in a V-formation. It is non-pathogenic and is

found in soil, animal feces, fruits and vegetables. Though it was originally isolated for its ability

to produce massive amounts of glutamic acid, C. glutamicum and closely related organisms have

been developed for the production of most of the biogene amino acids, nucleotides, and vitamins.

Because of this ability it has undergone extensive genetic study to understand its production

pathways.The isolation of C. glutamicum as a producer of glatamic acid and sequentially for the

large-scale production of MSG was achieved in post WWII Japan by Japanese researchers led by

S. Kinoshita at Kyowa Hakko Kogyo Co. They found soil bacteria that produced large amounts of

glutamic acid. They made two duplicate plates and after letting them grow for awhile, they killed

the bacterium on one plate by UV irradiation. They then inoculated that plate with Leuconostoc

mesenteroides, a bacteria that will only grow in the presence of glutamic acid. By looking at the

locations of the L. mesenteroides colonies and comparing to the duplicate plate, they isolated the

glutamic acid producing bacteria, C. glutamicum. Wild-type strains of this bacterium were found

to produce about 10 g/L glutamic acid, and with genetic engineering yields are now upwards of

100g/L. The thing that distinguishes C. glutamicum from similar bacteria that produce the glutamic

acid is the amount that is produced. Glutamic acid is made by extracting alpha-ketogluterate from

the TCA cycle by way a reductive amination by NADP+ specific glutamate dehydrogenase. The

reason that C. glutamicum produces so much more than similar bacteria is not known for sure. A

widely accepted theory used to be that C. glutamicum had very little alpha-ketogluterate

dehydrogenase, the enzyme needed to continue the TCA cycle, so it was forced to produce

glutamic acid.

Corynebacterium glutamicum

That was not necessarily disproved because of the difficulty of isolating alpha-ketogluterate

dehydragenase, but it is now thought to have more to do with concentrations of glutamic acid

inside the cell. In biotechnological processes, Corynebacterium species are used for economic

production of glutamic acid by submerged fermentation. L-glutamic acid is produced per year

using coryneform bacteria. A number of fermentation techniques have been used for the

production of glutamic acid. Glucose is one of the major carbon sources for production of glutamic

acid. Glutamic acid was produced with various kinds of raw materials using sub-merged

fermentation of palm waste hydrolysate, cassava starch, sugar cane bagasse, date waste.Properties

of a useful industrial microbe include

- Produces spores or can be easily inoculated.

- Grows rapidly on a large scale in inexpensive medium.

- Produces desired product quickly.

- Should not be pathogenic.

- Amenable to genetic manipulation.

Industrial production of glutamic acid

The manufacturing process of glutamic acid by fermentation comprises :- fermentation, crude

isolation, and purification processes.

The fermentation unit in industrial microbiology is analogous to a chemical plant in the chemical

industry. A fermentation process is a biological process and, therefore, has requirements of sterility

and use of cellular enzymic reactions instead of chemical reactions aided by inanimate catalysts,

sometimes operating at elevated temperature and pressure. Industrial fermentation processes may

be divided into two main types, with various combinations and modifications.

1- Batch culture

Batch fermentation widely use in production of most of amino acids. refers to a partially closed

system in which most of the materials required are loaded onto the fermentor, decontaminated

before the process starts and then, removed at the end. The only material added and removed during

the course of a batch fermentation is the gas exchange and pH control solutions. In this mode of

operation, conditions are continuously changing with time, and the fermentor is an unsteady-state

system, although in a well-mixed reactor, conditions are supposed to be uniform throughout the

reactor at any instant time. The principal disadvantage of batch processing is the high proportion

of unproductive time (down-time) between batches, comprising the charge and discharge of the

fermentor vessel, the cleaning, sterilization and re-start process

2- Fed-batch fermentation

The fed-batch technique was originally devised by yeast producers in the early 1900s to regulate

the growth in batch culture of Saccharomyces cerevisiae18. Yeast producers observed that in the

presence of high concentrations of malt, a by-product - ethanol - was produced, while in low

concentrations of malt, the yeast growth was restricted. The problem was then solved by a

controlled feeding regime, so that yeast growth remained substrate limited. The concept was then

extended to the production of other products, such as some enzymes, antibiotics, growth hormones,

microbial cells, vitamins, amino acids and other organic acids. Basically, cells are grown under a

batch regime for some time, usually until close to the end of the exponential growth phase. At this

point, the reactor is fed with a solution of substrates, without the removal of culture fluid. This

feed should be balanced enough to keep the growth of the microorganisms at a desired specific

growth rate and reducing simultaneously the production of by-products (that can be growth or

product production inhibitory and make the system not as effective). These by-products may also

affect the culture environment in such a way that might lead to early cell death even though

sufficient nutrients are available or are still being provided.

A fed-batch is useful in achieving high concentration of products as a result of high concentration

of cells for a relative large span of time. Two cases can be considered: the production of a growth

associated product and the production of a non-growth associated product. In the first case, it is

desirable to extend the growth phase as much as possible, minimizing the changes in the fermentor

as far as specific growth rate, production of the product of interest and avoiding the production of

by-products. For non-growth associated products, the fed-batch would be having two phases: a

growth phase in which the cells are grown to the required concentration and then a production

phase in which carbon source and other requirements for production are fed to the fermentor. This

case is also of particular interest for recombinant inducible systems: the cells are grown to high

concentrations and then induced to express the recombinant product. Also, considering that

plasmid stability is very often guaranteed by the presence of an antibiotic marker gene and that the

lifetime of this antibiotic in a fermentor can be limited, it might be of interest to use the fed-batch

concept to feed this same antibiotic continuously so that the presence of the plasmid in the cells is

more of a reliable fact. Fed-batch fermentations can be the best option for some systems in which

the nutrients or any other substrates are only sparingly soluble or are too toxic to add the whole

requirement for a batch process at the start, in fermentations such as mycelial culture the increase

of viscosity with time can be compensated by the addition of relatively small quantity of water

during the fermentation time, although the efficacy of this protocol is controversial among

reserachers. Many factors are involved in the regulation of a fed-batch reactor. As an example,

however, the feed rate can be varied to control the concentrations of nutrients in the bioreactor.

3- Continuous fermentation

Continuous culture is a technique involving feeding the microorganism used for the fermentation

with fresh nutrients and, at the same time, removing spent medium plus cells from the system1.

An unique feature of the continuous culture is that a time-independent steady-state can be attained

which enables one to determine the relations between microbial behavior (genetic and phenotypic

expression) and the environmental conditions.

Industrial production of glutamic acid

- Natural product such as sugar cane is used.

- Then, the sugar cane is squeezed to make molasses.

- The heat sterilize raw material and other nutrient are put in the tank of the fermenter.

- The microorganism (Corynebacterium glutamicum) producing glutamic acid is added to the

fermentation broth.

- The microorganism reacts with sugar to produce glutamic acid.

- Then, the fermentation broth is acidified and the glutamic acid is crystallized.

Separation and purification

- After the fermentation process, specific method is require to separate and purify the amino acid

produced from its contaminant products, which include:

Centrifugation. Common method used in industry, can be operate semi-continuous or continuous

basis, large scale tests have to performed to choose a suitable centrifuge, poor centrifugation can

be improved by adding flocculation agent. This agent will neutralize the anionic charges on the

surface of microbial cells.

Filtration. Also widely use in industrial, based on a few factors : Properties of the filtrate, nature

of the solid particles, Adequate pressure to obtain adequate flow rate, negative effects of

antifoaming agents on filtration, filtration can be improved by using filteraids, filteraids improved

the porosity of a resulting filter cake leading to a faster flow rates.

Crystallisation. Method to recover amino acid, because of the amphoteric character of amino acid,

their solubility are greatly influenced by the pH of a solution, temperature also influence the

solubility of amino acid and their salts, thus, lowering the temperature can be used to obtain the

required product, precipitation of amino acid with salts are commonly used

Ion exchange. Used for the extraction and purification of amino acids form the fermentation broth,

strongly affected by pH of the solutions and the present of contaminant ions. There are two types

of ion exchange resins, cation exchange resins, anion exchange resins. cation exchange resins, bind

with positively charged amino acids, Anion exchange resins, bind with negatively charged amino

acid. Anion exchange resins are generally lower in their exchange capacity and durability than

cation exchange resins, ion exchange as a tool for separation is only used when other steps fail,

because of its tedious operation, small capacity and high costs.

Electro-dialysis. Based on the principle that charged particles move towards the electrodes in the

electric field, a mixture of the required amino acid and contaminant salts can be separated at a pH

where the amino acid has a net zero charge. The salt ions are captured by the ion exchange

membranes that are present, the applications are limited to desalting amino acid solutions.

Solvent extraction. Has only limited applications. The distribution coefficients of amino acids

between organic solvent and water phases are generally small. Some possibilities based on

alteration of amino acid, cyclisation of L-glutamic acid and extraction with alkyl and aromatic

alcohols, conversion of contaminant organic acids (like acetic acid) to the ester form and extraction

of the ester, extraction of basic amino acids (like L-lysine) from aqueous solution with water

immiscible solvents containing higher fatty acids;

Decolorisation. Performed to get rid of the colored impurities in the fermentation broth. based on

the fact that amino acids (especially the non-aromatic amino acids) do not adsorb onto activated

charcoal. Although the treatment is very effective, some of the amino acid is lost during this step.

Alternative ways: addition of cationic surfactants, high molecular synthetic coagulants or some

phenolic compounds, washing of crystals with weakly alkaline water as in the case of glutamic

acid.

Evaporation. Evaporation of the amino acid containing solution is a quick but commercially

unattractive way (high energy costs) to obtain amino acids from solution, used when the total

amount of contaminant products is very low, since these compounds are not removed and appear

in a concentrated form in the product.

After undergo to the suitable method of separation and purification, the glutamic acid crystal cake

is then separated from the acidified fermentation broth. The glutamic acid crystal is added to the

sodium hydroxide solution and converted into monosodium glutamate (MSG), which is more

soluble in water, less likely absorb moisture and has strong umami taste. The MSG is cleaned by

using active carbon, which has many micro holes on their surface, the clean MSG solution is

concentrated by heating and the monosodium glutamate crystal is formed. The crystal produce are

dried with a hot air in a closed system. Then, the crystal is packed in the packaging and ready to

be sold.

Advantages of amino acid fermentation

- Normally the production strain is constructed in such a way that overproduction of the desired

amino acid is obtained and no, or only minor concentrations of, unwanted contaminants appear.

- Optical resolution steps are not necessary (as in the case of most chemical-processes) since

only the L-form is synthesised.

- The required amino acid can be relatively easily separated from cells and protein impurities.

References

1. Masato Ikeda, Microbial Production of l-Amino Acids Advances in Biochemical

Engineering/Biotechnology.Amino Acid Production Processes, 79, pp. 1-35, 2003.

2. Hidehiko Kumagai. Amino Acid Production the Prokaryotes 3rd edition, pp. 169-177, 2013.

3. Chibata, I., T. Kakimoto, and J. Kato. Enzymatic production of L-alanine by Pseudomonas

dachnae Appl. Microbiol. 13, pp. 638–645, 1965.

4. Sano K, Mitsugi K, Enzymatic production of L-cysteine from DL-2-amino-Δ2-thiazoline-4-

carboxylic acid by Pseudomonas thiazolinohilum: optimal conditions for the enzyme formation

and enzymatic reaction. Agric Biol Chem (42), pp. 2315–2321, 1978.

5. Sano K, Eguchi NY, Mitsugi K Metabolic pathway of L-cysteine formation from DL-2-amino-

Δ2-thiazoline- 4-carboxylic acid by Pseudomonas. Agric Biol Chem 43:2373–2374, (1979).

6. Terasawa M, Yukawa H, Takayama Y, Production of Laspartic acid from Brevibacterium by

the cell re-using process. Process Biochem 20:124–128, (1985).

7. Takamatsu S, Umemura I, Yamamoto K, Sato T, Chibata I, Production of L-alanine from

ammonium fumarate using two immobilized microorganisms. Eur J Appl Microbiol Biotechnol

15:147–152, (1982).

8. Srinivasan VR, Summers RJ, Continuous culture in the fermentation industry. In: Calcott PH

(ed) Continuous cultures of cells. CRC Press, Florida, 97, (1981).

9. Kinoshita S, Taxonomic position of glutamic acid producing bacteria. In: Flickinger MC, Drew

SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation.

Wiley, 1330, (1999).

10. Kinoshita S, Tanaka K, Glutamic acid. In: Yamada K (ed) The microbial production of amino

acids. Wiley, 263, (1972)

11. Y Anzai, H Kim, J Y Park, H Wakabayashi and H Oyaizu, Phylogenetic affiliation of the

pseudomonads based on 16S rRNA sequence. IJSEM, 50 (4): 1563- 1589, (2000).

12- Kimura, E., C. Yagoshi, Y. Kawahara, T. Ohsumi, T. Nakamatsu, and H. Tokuda, Glutamate

overproduction in Corynebacterium glutamicum triggered by a decrease in the level of a complex

comprising dtsR and a biotincontaining subunit Biosci. Biotechnol. Biochem. 63: 1274–1278,

(1999).

13. Yamada, H., and H. Kumagai, Synthesis of L-tyrosine related amino acids by β-tyrosinase In:

D. Perlman (Ed.) Advances in Applied Microbiology Academic Press New York NY 19: 249–

288, (1975).

14. Katsumata R, Mizukami T, Kikuchi Y, Kino K, Threonine production by the lysine producing

strain of Corynebacterium glutamicum with amplified threonine biosynthetic operon. Genetics of

industrial microorganisms, 21(1): 217, (1986).